Method of protein production in plants

ABSTRACT

A process of producing a protein or polypeptide of interest in a plant or in plant is provided, comprising: (i) transforming or transfecting a plant of plant cells with a nucleotide sequence having a coding region encoding a fusion protein comprising the protein or polypeptide of interest, a signal peptide functional for targeting said fusion protein to the apoplast, and a polypeptide capable of binding the fusion protein to a cell wall component, (ii) enriching cell wall components having expressed and bound fusion protein, and separating the protein or polypeptide of interest or a protein comprising the protein or polypeptide interest.

FIELD OF THE INVENTION

The present invention relates to a process and vectors for theproduction of a protein of interest in a plant or in plant cells andproteins and polypeptides obtained thereby. Further, the inventionrelates to vectors for this process and to plants or plant cellstransformed therewith.

BACKGROUND OF THE INVENTION

Recombinant protein production in plant systems has been very successfulfor many different products, covering proteins with industrialapplications, food and feed additives, animal health products and humanpharmaceuticals, such as antigens and immune response proteins.

There are many comprehensive reviews describing the field (Daniell, etal., 2001 Trends Plant Sci., 2001, 6:219-226; Larrick &Thomas, 2001,Curr. Opin. Biotech., 12:411-418; Doran, 2000, Curr. Opin. Biotech.,11:199-204; Hood & Jilka, 1999, Curr. Opin. Biotech., 10;382-386) Plantshave been considered as a low-cost production system for proteins, thatis significantly cheaper in comparison with bacterial, yeast, insect andmammalian cell-based production systems. However, little considerationwas given to the technical challenges associated with downstream proteinpurification from plant material. Recovery and purification of proteinsfrom biomass is an expensive and technically complex process, and it mayaccount for more than 90% of the total production cost, especially in“inexpensive” production systems such as yeasts or microbial cells.Purification from plant biomass is a priori even more difficult, and mayrepresent the most serious bottleneck on the way to industrialization ofplant-based production platforms. The available data in this regardconfirm the above said. For example, extraction of β-glucuronidase fromtransgenic corn seeds accounts for 94% of the production cost(Evangelista et al., 1998, Biotechnol. Prog., 14:607-614).

A number of processes have been specifically designed in order toaddress the problem. Compartmentalization of the recombinant proteinwithin the plant cell followed by its secretion is one pre-requisite ofmaking the product easily purifiable. Rhizosecretion of recombinantproteins was demonstrated for engineered tobacco plants (Borisjuk etal., 1999, Nat. Biotechnol., 17, 466469; US 6096546). It was shown thattargeting a protein to the endoplasmatic reticulum using a fusion with asignal peptide allows to obtain secreted protein that is alreadypartially purified (is in the secretion fluid, along with just a dozenor so of proteins normally secreted by a plant) and in significantlyhigher amounts, as opposed to isolating it from homogenized planttissues that are highly complex mixtures of thousands of proteins andother molecules. A similar approach was used with plant viral expressionvectors, where the protein was targeted to the apoplast so thatpurification begins with a highly enriched product (for review see:Yusibov et al., 1999, Curr Top. Microbial. Immunol., 240, 81-94).Another technology, developed by M. Moloney (U.S. Pat. No. 5,650,554),solves the problem by targeting the protein of interest, as a part ofoleosin fusion, to oil bodies, i.e. a cell fraction that is easilyremovable.

Yet another important strategy is to equip the recombinant protein withan affinity tag, thus facilitating the purification of proteins throughaffinity chromatography. This method is widely described for in vitropurification of proteins using various affinity tags, such aspolyhistidine, Flag, protein A, gluthatione-S-fransferase,cellulose-binding domains (CBD), etc. (Sassenfeld, 1990, TIBTECH, 8,88-93). In the case of CBD fusion proteins, the protein of interest isfused to a substrate-binding region of a polysaccharidase (cellulases,chitinases and amylases, as well as xylanases and the β-1,4 glycanases).The affinity matrix containing the substrate such as cellulose can beemployed to immobilize the polypeptide. The polypeptide or fusionprotein can be removed from the matrix using a protease cleavage site.Different variants of this approach using CBDs of plant and bacterialorigin are described in several publications and patents (Ong et al.,1991, Enzyme Microb. Technol., 13, 59-65; Linder et al., 1998,Biotechnol. Bioeng., 60, 642-647; U.S. Pat. No. 5,137,819; U.S. Pat. No.5,202,247; U.S. Pat. No. 5,670,623; U.S. Pat. No. 5,719,044; U.S. Pat.No. 5,962,289; U.S. Pat. No. 6,060,274). This version of purificationhas never been tested with plants, because of concerns that the materialof interest will be attached to the plant cell wall that containscellulose and will be impossible to purify. A further concern has beenthat targeting proteins to the plant cell wall interferes with plantgrowth (WO 00/77174).

The approaches described above focus either on targeting recombinantproteins to a secretory pathway or on employing affinity tags, such ascellulose binding domains for an in vitro purification process. Thereare also inventions describing the subcellular targeting of cellulolyticenzymes for large scale cellulase production and cellulose degradation(WO 9811235), or for generating transgenic plants with altered structureor morphology (U.S. Pat. No. 6,184,440). None of these patents focuseson improving the protein purification procedure. Rather, they addressproblems of cellulolytic enzyme toxicity for large-scale productionand/or their use for modulation of cell wall morphology. Actually, thesetechnologies are limited to the expression of cell wall degrading ormodifying enzymes. Of course, for the production of such enzymes inplants, a subcellular localization is preferred. However, cellulases andcell wall modifying enzymes represent only a small fraction of proteinswith commercial value. There is no technology for cheap large scaleproduction and purification of recombinant proteins in general,especially of cytotoxic proteins in plants.

U.S. Pat. No. 5,474,925 discloses a method of targeting a recombinantprotein of interest to cotton fibers in cotton plants. The protein ofinterest is immobilised on said cotton fibers and is used in industrialprocesses in this immobilised state.

Ziv & Shoseyov (U.S. Pat. No. 6,331,416, WO 00/77174) describe a methodof expressing a recombinant protein of interest as a protein fusion witha cellulose binding domain (CBD), whereby binding of said CBD to cellwall cellulose is used during work up for affinity purification of theprotein fusion. Since binding of a protein to cell wall celluloseinterferes with plant growth and leads to growth arrest, the authorscompartmentalise the fusion protein within cells in order to sequesterthe fusion protein from the cell walls. The protein fusions are broughtin contact with cell wall cellulose by homogenising the plant material.This methods has several severe disadvantages which render itpractically useless for large large scale or commercial applicationslike molecular farming:

-   (i) since the fusion protein is expressed during plant growth and    accumulate in cellular compartments, the plant suffers from a    substantial burden during growth. Thus, plant growth is slowed down    and the maximum plant size is reduced compared to the wild-type    plants leading to a loss of biomass and to limited amount of    produced recombinant protein. More plants would have to be grown to    achieve the same biomass as with wild-type plants meaning    consumption of more expensive greenhouse space more expenditure    during down-stream processing and protein purification;-   (ii) proteins that are toxic to the plant cannot be expressed;-   (iii) plants expressing a particular fusion protein have to be    raised starting from transgenic seeds or plantlets requiring    essentially a whole season for the production of a particular    protein of interest. The molecular farmer cannot react to short-term    orders for a particular protein. Therefore, this method is extremely    unflexible.

It is an object of the invention to provide a process of producing aprotein of interest in plants, whereby interference of foreign proteinexpression with plant growth is not a problem.

It is a further object of the invention to provide a process ofproducing a protein of interest in plants that allows production ofproteins that are toxic to the plant.

It is a further object of the invention to provide a process ofproducing a protein of interest in plants or plant cells, which issignificantly simpler compared to existing technologies and offers aneconomic way of protein production in plants.

GENERAL DESCRIPTION OF THE INVENTION

These objects are solved by a process of producing a protein orpolypeptide of interest in a plant or in plant cells, comprising:

-   (i) transforming or transfecting a plant or plant cells with a    nucleotide sequence having a coding region encoding a fusion protein    comprising the protein or polypeptide of interest, a signal peptide    functional for targeting said fusion protein to the apoplast, and a    polypeptide capable of binding the fusion protein to a cell wall    component,-   (ii) enriching cell wall components having expressed and bound    fusion protein, and-   (iii) separating the protein or polypeptide of interest or a protein    comprising the protein or polypeptide of interest.

The inventors of the present invention managed to overcome theshortcomings of the prior art by recognising that the life time of aplant used to express a protein of interest can be devided into twoconceptually different phases: (i) plant life and growth beforetransfomaton or transfection and (ii) plant life and growth aftertransformation or transfection.

In the process of the invention, a plant used for expressing said fusionprotein is preferably first grown to a desired growth state beforetransformation or transfection. Said desired growth state may be agrowth state which is favourable for the expression of a particularfusion protein. Said growth state is preferably close to the maximumgrowth the plant can achieve, i.e. when maximum or near maximum biomasshas accumulated. Transforming or transfecting a plant that hasaccumulated substantial biomass has the following advantages: (a) manycells and/or a big amount of biomass is available per plant for theexpression of the fusion protein; (b) binding of the fusion protein tothe cell wall in the apoplast does not interfere with plant growth orsuch interference does not impede the process of the invention; (c) thenucleotide sequence used for transformation can be selected at a latestage, providing a high degree of flexibility to the molecular farmer.

The approach of the invention has the essential advantage that plantgrowth is not perturbed by the expression of the fusion protein. Plants,notably wild type plants, can be grown up to a desired growth statewithout any burden by the expression of said fusion protein. Since themaximum amount protein of interest that can be produced depends on theplant biomass, more protein of interest can be produced per plant thanin prior art processes, where plant growth is impeded by the expressionof a foreign protein and/or accumulation of a foreign protein incompartments of the cells or in the cell wall. Therefore, the process ofthe invention is more efficient, more productive, and cheaper than priorart processes.

A plant having expressed said fusion protein may be harvested forcarrying out step (ii) at any suitable time. As described above,transforming or transfecting a grown plant is preferred according to theinvention, since the time required between transforming or transfectingand harvesting said plants can be reduced enormously. Plants are thenpreferably harvested soon after said transforming or transfecting. Morepreferably, plants are harvested between one and seven days, mostpreferably after two to four days after said transforming ortransfecting. In contrast to the prior art, disturbance of cell wallfunction by binding of the fusion protein is therefore not a problem.

Moreover, the process of the invention provides the molecular farmerwith a great degree of flexibility, since the decision of which proteinto express (which nucleic acid to transform) can be postponed until alate stage in plant development. If a molecular farmer has grown plantsat hand and a number of vectors each encoding a commercial protein, hecan react quickly to sudden market requirements and provide big amountsof a protein of interest on short notice, e.g. within one week. Theprocess of the invention therefore fits to modern market needs such asjust-in-time production. In contrast, prior art processes requirelong-term planning of the protein to be produced.

The process of producing a protein or polypeptide of interest accordingto the invention involves expression of said protein or polypeptide ofinterest as a fusion protein which is secreted from the cells whichexpress the fusion protein by way of a signal peptide. In the apoplast,the fusion protein will bind to a cell wall component by way of saidpolypeptide capable of binding the fusion protein to a cell wallcomponent. Thus, the plant cell wall or its components are exploited asaffinity matrix for purifying the protein or polypeptide of interest ora protein comprising the protein or polypeptide of interest.

In the first step of the process of the invention, a plant or plantcells are transformed or transfected with a nucleotide sequence having acoding region encoding said fusion protein. Transformation may producestably transformed plants or plant cells, e.g. transgenic plants.Alternatively, said plant or plant cells may be transfected fortransient expression of said fusion protein. Transient transfection ofgrown up plants is preferred.

Several transformation or transfection methods for plants or plant cellsare known in the art and include Agrobacterium-mediated transformation,particle bombardment, PEG-mediated protoplast transformation, viralinfection etc. For the preferred embodiment of transient expression oftransfection for transient expression, viral infection orAgrobacterium-mediated transformation advantageously employed.

Said nucleotide sequence may be DNA or RNA depending on thetransformation or transfection method. In most cases, it will be DNA. Inan important embodiment, however, transformation or transfection isperformed using RNA virus-based vectors, in which case said nucleotidesequence is RNA.

One very convenient way is to use a DNA vector that is based on a virus.Preferably, the DNA vector is based on an RNA virus, i.e. the DNAvectors contains the cDNA of RNA viral sequences in addition to saidnucleotide sequence. Examples of plant DNA or RNA viruses sequences ofwhich may be used for viral vectors according to the invention are givenin WO 02/29068 and in PCT/EP02/03476. Such DNA vectors further contain atranscriptional promoter for producing the RNA viral transcript. Inthese embodiments, transformation or transfection is preferably carriedout by viral transfection, more preferably via Agrobacterium-mediatedtransformation.

Said nucleotide sequence comprises a coding region encoding a fusionprotein. Said fusion protein comprises the protein or polypeptide ofinterest (referred to as “protein of interest” in the following). Saidprotein of interest may be any protein or polypeptide that can beproduced and isolated according to the process of the invention. It maybe produced in an unfolded, misfolded or in a natural, functionalfolding state. The latter possibility is preferred. Pharmaceuticalproteins are particularly preferred.

Said fusion protein further comprises a signal peptide functional fortargeting said fusion protein to the apoplast. This may be achieved witha signal peptide that targets the fusion protein into the endoplasmaticreticulum (ER) and the secretory pathway. All signal peptides ofproteins known to be secreted or targeted to the apoplast may be usedfor the purposes of the invention. Preferred examples are the signalpeptides of tobacco calreticulin or of rice amylase. Further examplesare the signal peptides of pectin methylesterase or of N. tabaccotyrosine and lysine rich protein (NtTLPR). A further example is thesignal peptide of apoplastic isoperoxidase from zucchini (APRX) (Carpinet al., 2001, The Plant Cell, 13, 511-520). The signal peptide has to becomprised in said fusion protein such that it is functional for saidtargeting. This conditions may be fulfilled at any location in thefusion protein. In general, however, the signal peptide is positioned atthe N-terminus of the fusion protein for functional targeting of thefusion protein to the apoplast.

Said fusion protein further comprises a polypeptide capable of bindingthe fusion protein to a cell wall component. By way of said polypeptide,a predominant fraction of the fusion protein secreted to the apoplast isbound or immobilized on the cell wall. Binding to the cell wall may beto any cell wall component. Cell wall components for the purposes ofthis invention include polysaccharides like cellulose, hemicellulose,β-1,4-glycan, pectin etc. and non-polysaccharides like proteins orlignin. Binding to polysaccharide components of the cell wall ispreferred. Most preferred is binding to cellulose.

Pectin is another major cell wall polymer found in all land plants(Willats et al., 2001, Plant Mol Biol, 47, 9-27). Pectin is a complexpolymeric network of polysaccharides like homogalacturonan andrhamnogalacturonan. The main monomeric component of this network isgalacturonic acid and not glucose like in the case of cellulose.Peroxidases are involved in the construction of cell walls and in thecontrol of cell wall plasticity by catalysing the crosslinking ofaromatic molecules using hydrogen peroxide as an electron acceptor.Another reaction known to be catalysed by peroxidases is theestablishment of covalent bonds between hydroxycinnamate ester moietiesor flavonoids associated with pectins or hemicellulose. For severalperoxidases a specific interaction between the cell wall polymerhomogalacturonan was found which seems to be essential for theiractivity. In one example of this invention we have used Ca²⁺-pectatebinding site of apoplastic isoperoxidase from zucchini (APRX) (Carpin etal., 2001, The Plant Cell, 13, 511-520). Said site binds in vivo as wellin vitro to pectin chains through cross-links formed by Ca²⁺-ions (seeExamples 6 and 7).

Examples of polypeptides capable of binding the fusion protein to a cellwall component are proteins which use such a cell wall component as asubstrate like cellulases or hemicellulases. The substrate bindingdomain of such enzymes is preferably used as said polypeptide capable ofbinding. Further examples are polysaccharide binding domains (PBDs) ofmicrobial origin (see below). An example specifically exemplified hereinis pectin methylesterase (Examples 1 to 3). In these examples, bindingto a cell wall component will in general be non-covalent. Alternatively,binding may involve a covalent bond to a cell wall component(cross-linking). An example is NtTLPR which can form disulfide bonds toa proteinaceous cell wall component.

In step (ii) of the process of the invention, cell wall componentshaving expressed and bound fusion protein are enriched. This stepinvolves the removal of many compounds contained in plants and resultsin a substantial purification of the protein of interest to be produced.While secretion of the fusion protein out of the expressing cell(protoplast) separates the fusion protein containing the protein ofinterest spatially from most protoplast compounds, these protoplastcompounds may be removed in this step. Cell wail preparation proceduresknown in the art may be used in step (ii). Step (ii) may e.g. beperformed by harvesting the plant or plant cells followed byhomogenization of the plant tissue which contains expressed fusionprotein of the invention. Liquid and solid phases of the homogenisateshould then be separated e.g. by employing centrifugation or filtration.The cell wall components may also be enriched by pressing or squashinge.g. between rolls. The solid residue having the fusion protein bound orimmobilized thereto is then preferably washed with a suitable washingbuffer in order to remove soluble contaminants, again followed byrecovering the insoluble cell wall material. The conditions used in step(ii) are preferably chosen to preserve the folding state of the proteinof interest-domain in the fusion protein. The washing buffer ispreferably aqueous. However, use of organic solvents may also beadvantageous depending on the protein to be produced. In order to fullyexploit the advantages of the invention, the cell wall componentcarrying the fusion protein should stay insoluble, preferably at leastin the initial stages of the enriching of step (ii). However, theenriching/purification may be designed in many different ways and mayalso involve separation of cell wall components.

In the final step (iii) of the Invention, the protein of interest or afusion protein comprising the protein of interest is separated from thecell wall component to which it bound in step (i). This separation maybe done in many different ways. In one embodiment, the fusion proteinmay be separated from the cell wall or respective cell wall component.(It is noted that, in most cases, the fusion protein will not comprisethe signal peptide any more due to natural cleaving off during secretionof the fusion protein.) The way this may be done depends on thesituation, especially on the polypeptide used for binding the fusionprotein to the cell wall component. For many polypeptides which bind toa cell wall component, the conditions under which tight or loose bindingoccurs are known. Alternatively, these conditions can be determinedexperimentally. If the binding is non-covalent, binding may e.g. bereleaved or weakened by adjusting the ionic strength, the pH,temperature or a buffer component (e.g. a chaotropic agent) (Greenwoodet al. (1988) FEBS Lett. 224, 127-131; Shoseyov et al. (1992) Proc.Natl. Acad. Sci. USA 89, 3483-3487). In the case of covalent binding,the type of the covalent bond has to be taken into account. In the caseof NtTLRP as a polypeptide capable of binding to a cell wall component,addition of a reducing agent containing free —SH groups likemercaptoethanol or dithiothreitol for cleaving disulfide bonds may beused.

Preferably, the separation of step (iii) involves cleavage of the fusionprotein such that the protein of interest is obtained free oressentially free of other fusion protein components. This embodimentinvolves cleavage of at least one peptide bond. To this end, the fusionprotein may further comprise a cleavage sequence allowing cleavage ofthe fusion protein. The cleavage sequence may be a peptide recognized bya site-specific protease. Addition of the appropriate protease may thencut the protein of interest out of the fusion protein still bound to acell wall component. Removal of remaining cell wall components thenproduces the protein of interest free of most plant material. Ascleavage sequence/protease systems those generally used for removingaffinity tags from affinity-purified proteins may also be used for thisinvention. Such systems are commercially available e.g. from AmershamPharmacia Biotech, Uppsala, Sweden. A specific example frequently usedfor removing a His-tag is the factor Xa system. In another embodiment,ubiquitin may be included in the fusion protein and cleavage may beobtained with a ubiquitin-specific hydrolase. The cleavage sequence mayalso be autocatalytic, whereby cleavage may be achieved by providingappropriate conditions for cleavage, e.g. intein-mediated cleavage maybe used. The protein of interest thus obtained may be concentrated.Depending on the situation, the protein of interest may be subjected tofurther purification.

Construction of the nucleotide sequence of the invention may be doneaccording to standard procedures of molecular biology. Said nucleotidesequence preferably contains a plant-specific promoter operably linkedto said coding sequence and a transcription terminator after said codingregion. Tissue-specific expression of said fusion protein may beachieved if desired by appropriate selection of the promoter. In apreferred embodiment, virus-based vectors are used for performing thisinvention. Construction of such vectors is described in the referenceexamples.

Further information on practical aspects of step (ii) and (iii) of theinvention are found in WO 00/77174 references cited therein.

The nucleotide sequence comprises a coding region which codes for saidfusion protein. The components of said fusion protein may be arranged indifferent orders. However, the signal peptide is preferably placed atthe N-terminal end of the fusion protein in order to be functional fortargeting. The protein of interest is preferably placed at theC-terminus of the fusion protein, which allows its separation from theremainder of the fusion protein by a single peptide bond cleavage. Thepolypeptide capable of binding will then be located in between thesignal peptide and the protein of interest. However, different ordersare also comprised by the invention e.g. placing the polypeptide capableof binding to a cell wall component at the C-terminus of the fusionprotein. The protein of interest may further be flanked on both sides bya polypeptide capable of binding to a cell wall component, whereby thesepolypeptides may be the same or different. The protein of interest maythen be cleaved out by two proteolytic events. Preferably, these twoproteolytic events use the same cleavage sequence. The protein ofinterest may further be included in an intein for convenient isolation.

PREFERRED EMBODIMENTS OF THE INVENTION

In a preferred embodiment, this invention provides a process ofproducing a protein or polypeptide of interest in a plant, comprising:

-   (ia) growing a plant to a desired growth state;-   (ib) transiently transforming or transfecting the plant of step (ia)    or cells thereof with a viral vector, said vector containing a    nucleotide sequence having a coding region encoding a fusion protein    comprising the protein or polypeptide of interest, a signal peptide    functional for targeting said fusion protein to the apoplast, and a    polypeptide capable of binding the fusion protein to a cell wall    component,-   (ii) enriching cell wall components having expressed and bound    fusion protein, and-   (iii) separating the protein or polypeptide of interest or a protein    comprising the protein or polypeptide of interest,    whereby said polypeptide capable of binding the fusion protein to a    cell wall component binds to cellulose or to pectin; binding to    pectin is most preferred.

In another preferred embodiment, this invention provides a process ofproducing a protein or polypeptide of interest in a plant, comprising:

-   (i) transforming or transfecting the plant or cells thereof with a    nucleotide sequence having a coding region encoding a fusion protein    comprising the protein or polypeptide of interest, a signal peptide    functional for targeting said fusion protein to the apoplast, and a    polypeptide capable of binding the fusion protein to a cell wall    component,-   (ii) enriching cell wall components having expressed and bound    fusion protein, and-   (iii) separating the protein or polypeptide of interest or a protein    comprising the protein or polypeptide of interest,    whereby said nucleotide sequence is amplified and/or expressed in    said plant by a process comprising: providing the plant or cells    thereof with at least one precursor vector designed for undergoing    processing in cells of said plant, whereby due to said processing    said plant cell is endowed with at least one replicon which provides    for said amplification and/or expression.

More preferably, the plant or cells thereof is provided with at leasttwo precursor vectors. Alternatively, due to said processing, said plantcell is endowed with at least two replicons which

-   (a) are structurally related to each other owing to said processing;-   (b) are functionally distinct from each other; and-   (c) provide for said amplification and/or expression.    The embodiments involving precursor vectors are further described    below and are exemplified in example 6 and 7.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the general scheme of recombinant protein production inplant cells according to the invention.

-   -   A—transgene delivery into the plant cells by different means        (viral vectors —RNA, DNA viruses; stable nuclear or plastid        transformation; transient Agrobacterium-mediated        expression—“agroinfiltration”);    -   B—recombinant protein synthesis and attachment to cell wall;    -   C—enrichment of the plant tissue for recombinant protein by        squashing-out cytosol;    -   D—separation of recombinant protein from the plant tissue.

FIG. 2 shows the cDNA sequence of the full tobacco pectin methylesterase(PME) gene. Translation start and stop codons are shown in bold.

FIG. 3 shows an alignment of the deduced amino acid sequences of PMEgenes from tobacco (accession No. AJ401158), tomato (accession No.U49330) and orange (accession No. U82976). Alignment of deduced aminoacid sequences was performed using the Genebee program package. Thearrow indicates the N-terminus of a processed PME protein. Thetransmembran helix (black box) and PME signatures 1 and 2 are boxed.

FIG. 4 depicts the cloning schemes for different versions of PME-GFPfusions:

-   -   (A) cloning scheme for vectors plC2452 (full-length PME-GFP) and        plC2462 (mature PME);    -   (B) cloning scheme of vectors plC2472 (GFP-full-length PME) and        plC2482 (GFP-mature PME.

FIG. 5 shows in vitro RRL (rabbit reticulocyte lysate) translation offour GFP fusions with the PME gene. Lane 1: plC2482, lane 2: plC2452,lane 3: plC2472, and lane 4: plC2462.

FIG. 6 depcits the cloning scheme of different GFP-PME fusions in aplant expression cassette for transient expression experiments.

FIG. 7 shows N. benthamiana cells expressing GFP (light areas) targetedinto the cell wall after bombardment with a full length PME-GFP fusionunder the control of 35S promoter.

FIG. 8 depicts the T-DNA region of a binary vector carrying theflPME-GFP fusion.

FIG. 9 depicts a T-DNA region of a binary vector carrying NtTLPR-GFPfusion.

FIG. 10 depicts a T-DNA region of a binary vector carrying NtTLPRubq-GFPfusion.

FIG. 11 depicts a crTMV vector carrying NtTLPR-GFP fusion.

FIG. 12 depicts vectors T7/crTMV and SP6/crTMV.

FIG. 13 depicts vectors T7/crTMV/IRES_(MP,75) ^(CR)-GUS,T7/crTMV/IRES_(MP,75) ^(UI)-GUS, T7/crTMV/IRES_(MP,228) ^(CR)-GUS,T7/crTMV/IRES_(CP,148) ^(CR)-GUS, T7/crTMV/SPACER_(CP,148) ^(UI)-GUS andT7/crTMV/PL-GUS.

FIG. 14 depicts vectors plCH8600-C and plCH9666-C. IRES_(MP,75) ^(CR)stands for the internal ribosome entry site element of the movementprotein of a crucifer-infecting tobamovirus; see WO 02/29068 fordetails.

FIG. 15 depicts T-DNA-based vectors plCH4851 that provides the 5′-end ofthe viral pro-vector system, and plCP1010 that provides thesite-specific integrase PhiC31 mediating recombination between attP(plCP4851) and attB (plCP9666-C, FIG. 14) sites. Cloning of thesevectors is described in PCT/EP02/03476 and in FIG. 17.

FIG. 16 depicts vectors plCP9666-RIA and plCP9666-RI designed to expressthe restriction endonuclease EcORI.

FIG. 17 depicts the structure of a PVX-based precursor vector, itsgeneration by transcription (from plC3242) to give the complete form ofthe transcript and its splicing product. Expression can occur from thetranscript (precursor vector and replicon) and the spliced transcript(replicon).

FIG. 18 depicts basic constructs and cloning strategy used for cloningof the cDNA of a PVX-based precursor vector.

FIG. 19 depicts the general scheme of expression of two genes (CP andGFP) using a CrTMV-based spliceable precursor vector (provector).

FIG. 20 depicts the cloning strategy of intermediate construct plC3342.

FIG. 21 depicts the cloning strategy of intermediate constructs plC3378and plC3382.

FIG. 22 depicts the final stages of cloning plasmid plC3393.

FIG. 23 depicts the general scheme of expression of several genes viaCrTMV-based precursor vectors (a-c and vector shown at the top) andgeneration of replicons (A-C) by site-specific recombination atLoxP-sites catalyzed by the enzyme Cre recombinase.

FIG. 24 depicts the cloning scheme of construct plC3461—a primarycomponent of a recombination system.

FIG. 25 depicts the cloning scheme of construct plC3441—one of thesecondary components of a recombination system

FIG. 26 depicts the cloning scheme of construct plC3491—one of thesecondary components of a recombination system.

FIG. 27 depicts the cloning scheme of construct plC3521—one of thesecondary components of a recombination system.

FIG. 28 depicts the principal scheme of transgene expression via anoncontagious viral vector.

FIG. 29 depicts the structure of plasmid plC1593 used to introduce a Crerecombinase gene into the genome of N. benthamiana.

FIG. 30 depicts the general scheme of expression of several genes viaCrTMV-based precursor vectors (a-c and vector shown at the top) andgeneration of replicons (A-C) by using the integrase/att-system forsite-specific recombination. The precursor vectors are cloned intobinary vectors with T-DNA-borders (LB and RB).

FIG. 31 depicts the cloning scheme of construct plCP4851—one of thesecondary components of the recombination system.

FIG. 32 depicts the cloning scheme of construct plCH5151—one of thesecondary components of the recombination system.

FIG. 33 depicts the cloning scheme of construct plCH5161—one of thesecondary components of the recombination system.

FIG. 34 depicts the cloning scheme of construct plCH5951—one of thesecondary components of the recombination system.

FIG. 35 depicts the cloning scheme of the constructs plCH6871 andplCH6891—two of the secondary components of the recombination system.

FIG. 36 depicts the cloning scheme of construct plCH4371—one of thesecondary components of the recombination system.

FIG. 37 depicts the cloning scheme of plasmid plCH4461—one of thesecondary components of the recombination system.

DETAILED DESCRIPTION OF THE INVENTION

The general principle of the invention is shown in FIG. 1. A geneencoding a protein fusion with a secretory signal peptide and a cellwall binding domain is delivered into the plant cell preferably using aDNA or an RNA vector. The recombinant protein is expressed, targeted tothe intercellular space (apoplast) due to the presence of secretionsignal peptide and bound to the cell wall through its cell wall bindingdomain. The plants with said fusion protein are then subjected toprocessing (squashing or homogenization) in order to separate the cellwall matrix from the cell content. The cell wall matrix can then betreated in a way that allows separation of the recombinant protein fromsaid matrix.

Various methods can be used to deliver DNA or RNA vector into the plantcell, including direct introduction of said vector into a plant cell bymeans of microprojectile bombardment, electroporation or PEG-mediatedtreatment of protoplasts (for review see: Gelvin, S. B., 1998, Curr.Opin. Biotechnol., 9, 227-232; Hansen & Wright, 1999, Trends Plant Sci.,4, 226-231). Plant RNA and DNA viruses also present efficient deliverysystems (Hayes et al., 1988, Nature, 334, 179-182; Palmer et al., 1999,Arch. Virol., 144, 1345-1360; Lindbo et al., 2001, Curr. Opin. Plant.Biol., 4, 181-185). Said vectors can deliver a transgene either forstable integration into the genome/plastome of the plant (direct orAgrobacterium-mediated DNA integration) or for transient expression ofthe transgene (“agroinfiltration”).

The nucleic acid having the coding region coding for the protein ofinterest with a secretory signal peptide and a cell wall binding domaincan be designed in many different ways. Usually, the signal peptide islocalized at the N-terminal end of the fusion protein. The cell wallbinding domain(s) can link the signal peptide with the protein ofinterest or can be located at the C-terminal end of said fusion protein,or two cell wall binding domains may surround the protein of interest.In one embodiment of this invention, the whole tobacco pectinmethylesterase (PME cDNA, see FIG. 2; PME protein precursors, see FIG.3) gene was fused with a green fluorescent protein (GFP) gene as aprotein of interest. Pectin methylesterase (PME) (Gaffe et al., 1997,Plant Physiol., 114, 1547-1556; Dorokhov et al., 1999, FEBS Left., 461,223-228) is a secretory protein, which can be detected in theendoplasmatic reticulum (ER) and the cell wall. The members of the PMEmultigene family that undergo post-translational processing (Gaffe etal., 1997, Plant Physiol., 114, 1547-1556), are involved in cell wallturnover and appear to have a role in plant growth and development (Wenet al., 1999, Plant Cell, 11, 1129-1140). PME is known to be aubiquitous enzyme in the plant kingdom and regulates the cell walldegradation by catalyzing the demethoxylation of pectins. Mature 33 kDaPME is localized in the cell wall, while PME precursor is synthesized as70 kDa polyprotein. It is obvious that PME processing and trafficking tocell wall is accompanied by the removal of the N-terminal leadersequence, whereas anchoring of PME in the cell wall requires thespecific pectin binding domain.

In another embodiment of this invention (see examples 4 and 5) thefusion of a protein of interest was performed with the small (106 aminoacid residues) cell wall protein NtTLPR from tobacco, possessing asignal peptide sequence and a cysteine domain (CD). The CD is involvedin cross-linking the protein to the cell wall (Domingo et al., 1999,Plant J., 20, 563-570).

The fusion protein of the invention or said protein or polypeptide ofinterest may further contain an affinity peptide tag. Said affinitypeptide tag may be one of the following group: an oleosin protein orpart thereof, an intein or part thereof, an additional cell wall bindingdomain, a starch binding domain, a streptavidin epitope-tag sequence,glutathione-S-transferase (GST) affinity tag, a 6×His affinity tag, acalmodulin binding peptide (CBP) affinity tag. If said affinity peptidetag is an additional cell wall binding domain, it may be used forincreasing the binding affinity and/or likelihood of binding of thefusion protein to the cell wall. If more than one polypeptide capable ofbinding to a cell wall component is used, they may bind to the same orto different cell wall components. Other affinity peptide tags may beused to provide the fusion protein or the protein of interest withuseful properties, e.g. for (affinity) purification or for the use ofthe protein of interest.

The protein of interest can be fused with a signal sequence and cellwall binding domains from different genes, even from differentorganisms, for example an ER signal peptide of plant origin (e.g.tobacco calreticulin signal peptide) and a polisaccharide binding domain(PBD) of bacterial origin may be used in said fusion protein. Some plantpathogens (bacteria and fungi) are able to utilize polysaccharides suchas cellulose, pectin, xylan and several other carbon sources for theirgrowth and synthesize enzymes containing PBDs. The cellulose bindingdomains of said enzymes are well characterized and are extensively usedfor protein purification on a cellulose matrix. The affinity of aparticular cellulose or polysaccharide binding domain to a respectivecell wall component depends on the particular domain. Elution conditionsof a particular fusion protein having a particular CBD or PBD arepreferably established experimentally. CBDs usable for this inventionare e.g. those listed in U.S. Pat. No. 6,174,700.

Considering that recombinant protein production in this inventionincludes the fusion of said protein of interest with a signaling peptideand cell wall binding domain(s), the precise separation of the proteinof interest from the fusion protein can be an important issue. Vectorsthat include proteolytic sites flanking the coding sequence of interestwill allow to cleave the fusion protein and release the protein ofinterest in a pure form, if the conditions are provided that allow forsuch proteolytic cleavage. Such cleavage will be of specific advantage,if the cell wall binding domain in the fusion protein shows strongbinding and low recovery of fusion protein from the cell wall matrix. Itis observed with cell wall binding domain of the NtTLPR protein. Suchcleavage is of particular advantage, if the cell wall binding domain inthe fusion protein shows strong binding, which may result in a lowrecovery of the fusion protein from the cell wall matrix. This is e.g.observed with the cell wall binding domain of NtTLPR protein Cleavage ofa translational fusion protein can be achieved via a peptide sequencerecognized by a viral site-specific protease or via a catalytic peptide(Doija et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10208-10212;Gopinath et al., 2000, Virology, 267, 159-173; US5162601; US5766885;US5491076). Other examples of site-specific proteases applicable to thisinvention are mammalian enterokinases, for example, human enterokinaselight chain, which recognizes the sequence DDDK-I (Kitamoto et al.,1994, Prtoc. Natl Acad. Sci., 91, 7588-7592), and specifically cleavesLys-Ile bonds; viral proteases, like Hc-Pro (Carrington JC & Herndon KL, 1992, Virology, 187, 308-315) which catalyses proteolysis between theGly-Gly dipeptide but requires 4 amino acids for the recognition of thecleavage site; site-specific protease of Semliki Forest Virus (Vasiljevaet al., 2001, J. Biol. Chem., 276, 30786-30793); and proteases involvedin polyubiquitin processing, ubiquitin-carboxy-terminal hydrolases(Osava et al., 2001, Biochem Biophys Res Commun., 283, 627-633).

Ubiquitin-specific proteases open another opportunity. Expressingproteins and polypeptides as fusions to ubiquitin offers the advantageof an often-dramatic increase in expression level, and the ability toproduce any desired amino-terminal residue upon ubiquitin cleavage. Therecent availability of cloned ubiquitin-cleaving enzymes has enhancedthis technique for both bacterial and eukaryotic host systems (forreview see Baker, R. T., 1996, Curr. Opin. Biotech., 7, 541-546).Ubiquitin fusion expression systems are already described for plants(U.S. Pat. No. 5,773,705), yeasts (U.S. Pat. No. 6,068,994) and bacteria(U.S. Pat. No. 545,905). In one embodiment of this invention, theubiquitin polypeptide is inserted between the N-terminal NtTLPR proteinand the GFP gene (FIG. 10, Example 4). This allows to have a naturallyocuring N-terminal end of any protein of interest after cleavage of theubiquitin molecule by a ubiquitin-specific protease. The preciseness ofsuch cleavage does not depend on the N-terminal amino acid sequence ofthe protein of interest.

The gene of interest can be delivered into plants or plant cells usingviral vectors. Such an approach has an obvious advantage over theconstitutive expression of a transgene in stably transformed plants dueto the significantly higher expression level and independence fromcytotoxic effect, which might be caused by high level of transgenicproduct. The viral delivery system for this invention is described inExample 5 (FIG. 11) and 6 (FIGS. 14 to 16). The NtTLPR-GFP fusion isinserted into a crTMV expression vector. The apoplast targeted EcORIendonuclease-Ca²⁺-pectate binding site fusion is inserted into the 3′end of a TMV-based provector (Example 6). The use of these systems inplants is described in the reference examples 1 to 3 and in WO 02/29068.

The genes of interest that can be expressed and isolated using ourinvention include, but are not limited to: starch modifying enzymes(starch synthase, starch phosphorylation enzyme, debranching enzyme,starch branching enzyme, starch branching enzyme 11, granule boundstarch synthase), sucrose phosphate synthase, sucrose phosphorylase,polygalacturonase, polyfructan sucrase, ADP glucose pyrophosphorylase,cyclodextrin glycosyltransferase, fructosyl transferase, glycogensynthase, pectin esterase, aprotinin, avidin, bacterial levansucrase, E.coli gIgA protein, MAPK4 and orthologues, nitrogenassimilation/methabolism enzyme, glutamine synthase, plant osmotin, 2Salbumin, thaumatin, site-specific recombinase/integrase (FLP, Cre, Rrecombinase, Int, SSVI Integrase R, Integrase phiC31, or an activefragment or variant thereof), isopentenyl transferase, Sca M5 (soybeancalmodulin), coleopteran type toxin or an insecticidally activefragment, ubiquitin conjugating enzyme (E2) fusion proteins, enzymesthat metabolise lipids, amino acids, sugars, nucleic acids andpolysaccharides, superoxide dismutase, inactive proenzyme form of aprotease, plant protein toxins, traits altering fiber in fiber producingplants, Coleopteran active toxin from Bacillus thuringiensis (Bt2 toxin,insecticidal crystal protein (ICP), CryIC toxin, delta endotoxin,polyopeptide toxin, protoxin etc.), insect specific toxin AaIT,cellulose degrading enzymes, E1 cellulase from Acidothermuscelluloticus, lignin modifying enzymes, cinnamoyl alcohol dehydrogenase,trehalose-6-phosphate synthase, enzymes of cytokinin metabolic pathway,HMG-CoA reductase, E. coli inorganic pyrophosphatase, seed storageprotein, Erwinia herbicola lycopen synthase, ACC oxidase, pTOM36 encodedprotein, phytase, ketohydrolase, acetoacetyl CoA reductase, PHB(polyhydroxybutanoate) synthase, acyl carrier protein, napin, EA9,non-higher plant phytoene synthase, pTOM5 encoded protein, ETR (ethylenereceptor), plastidic pyruvate phosphate dikinase, nematode-inducibletransmembrane pore protein, trait enhancing photosynthetic or plastidfunction of the plant cell, stilbene synthase, an enzyme capable ofhydroxylating phenols, catechol dioxygenase, catechol 2,3-dioxygenase,chloromuconate cycloisomerase, anthranilate synthase, Brassica AGL15protein, fructose 1,6-biphosphatase (FBPase), AMV RNA3, PVY replicase,PLRV replicase, potyvirus coat protein, CMV coat protein, TMV coatprotein, luteovirus replicase, MDMV messenger RNA, mutant geminiviralreplicase, Umbellularia californica C12:0 preferring acyl-ACPthioesterase, plant C10 or C12:0 preferring acyl-ACP thioesterase, C14:0preferring acyl-ACP thioesterase (luxD), plant synthase factor A, plantsynthase factor B, Δ6-desaturase, protein having an enzymatic activityin the peroxysomal β-oxidation of fatty acids in plant cells, acyl-CoAoxidase, 3-ketoacyl-CoA thiolase, lipase, maize acetyl-CoA-carboxylase,5-enolpyruvylshikimate-3-phosphate synthase (EPSP), phosphinothricinacetyl transferase (BAR, PAT), CP4 protein, ACC deaminase, proteinhaving posttranslational cleavage site, DHPS gene conferring sulfonamideresistance, bacterial nitrilase, 2,4D monooxygenase, acetolactatesynthase or acetohydroxyacid synthase (ALS, AHAS), polygalacturonase,Taq polymerase, bacterial nitrilase, many other enzymes of bacterial orphage including restriction endonucleases, methylases, DNA and RNAligases, DNA and RNA polymerases, reverse trascryptases, nucleases(Dnases and RNAses), phosphatases, transferases etc.

Our invention also can be used for the purpose of molecular farming andpurification of commercially valuable and pharmaceutically importantproteins including industrial enzymes (cellulases, lipases, proteases,phytases etc.). Any human or animal protein can be expressed andpurified using described in our invention approach. Examples of suchproteins of interest include inter alia immune response proteins(monoclonal antibodies, single chain antibodies, T cell receptors etc.),antigens, colony stimulating factors, relaxins, polypeptide hormonesincluding somatotropin (HGH) and proinsulin, cytokines and theirreceptors, interferons, growth factors and coagulation factors,enzymatically active lysosomal enzyme, fibrinolytic polypeptides, bloodclotting factors, trypsinogen, α1-antitrypsin (AAT), human serumalbumin, native cholera toxin B as well as function-conservativeproteins like fusions, mutant versions and synthetic derivatives of theabove proteins.

Host plants for practicing this invention may be monocotyledonous ordicotyledonous plants. The most preferred plants include Nicotianaspecies including N. tabacum, Brassicacea species including canola,potato, maize, wheat, oat, rice, sugar beet, soybean, Pennisetum,cotton, alfa-alfa, sunflower, hemp, pea, carrot, cucumber, tomato, etc.Practically, any plant species with established transformation and/ortransfection protocol can be used in this invention.

Producing a Protein or Polypeptide of Interest Using Precursor Vectors(Pro-Vector System)

The present invention can easily be combined with other techniques ofplant engineering. A preferred example of such a technique is the“Pro-vector system” (PCT/EP02/03476). Examples 6 and 7 demonstrate thecombination of the present invention with precursor vectors(pro-vectors). Processes of causing amplification and/or expression ofone or more nucleic acid sequences of interest in a plant usingprecursor vector(s), which may be combined with the present invention,are described in the following.

A process of causing amplification and/or expression of one or morenucleic acid sequences of interest in a plant, plant tissue, plant cellor cell culture, characterized in that a plant cell is provided with atleast one precursor vector designed for undergoing processing in saidcell, whereby due to said processing said plant cell is endowed with atleast one replicon which provides for said amplification and/orexpression, whereby said at least one replicon is preferablystructurally related to each of said at least one precursor vectorsowing to said processing.

A process of causing amplification and/or expression of one or morenucleic acid sequences of interest in a plant, plant tissue, plant cellor cell culture, characterized in that a plant cell is provided with atleast one precursor vector designed for undergoing processing in saidcell, whereby due to said processing said plant cell is endowed with atleast two replicons which

-   (a) are structurally related to each other owing to said processing;-   (b) are functionally distinct from each other; and-   (c) provide for said amplification and/or expression.

A process of causing amplification and/or expression of one or morenucleic acid sequences of interest in a plant, plant tissue, plant cellor cell, culture, characterized in that a plant cell is provided with atleast two precursor vectors designed for undergoing processing in saidcell preferably by site-specific recombination, whereby due to saidprocessing said plant cell is endowed with at least one replicon whichprovides for said amplification and/or expression. Preferably, said atleast one replicon is structurally related to each of said at least twoprecursor vectors owing to said processing.

Further a process is described for the production of a biochemicalproduct, a process for gene function determination, and a process forartificial or directed evolution, whereby each of these processescomprises one of the above processes of causing amplification and/orexpression.

Moreover, vectors or precursor vectors and viral material for thisprocess are provided and viral material, replicons and plant materialobtained or obtainable by performing this process. Viral materialcomprises nucleic acids capable of replicating in a plant cell. Itcomprises infectious DNA or RNA. Viral material may be naked or coatedwith a coat protein.

Further, a kit-of-parts comprising (i) plant cells, seeds or plants and(ii) the above vectors, precursor vectors, viral material, or repliconsare provided. A further kit-of-parts is provided comprising (i) plantcells, seeds or plants and (ii) Agrobacterium cells containing the abovevectors, precursor vectors, viral material, or replicons.

The pro-vector process causes amplification and/or expression of one ormore nucleic acid sequences of interest in a plant cell. Amplificationrefers to the production of DNA or RNA (e.g. for anti-sense technology).Expression refers to the formation of a polypeptide or protein. In bothcases, the ultimate goal may be a biochemical product the production ofwhich may require amplification of said DNA or RNA and/or expression ofa polypeptide or protein.

The pro-vector process may be carried out in a plant, plant tissue,plant cell or cell culture. It is preferred to carry out said process inplant cells. Most preferably, said process is carried out in a plant.

Providing a plant cell with a precursor vector may comprise viraltransfection, Agrobacterium-mediated delivery, non-biological delivery,or conversion of a replicon pre-precursor DNA that was pre-integratedinto a plant nuclear DNA to form a precursor vector or vectors. However,said providing a plant cell with a precursor vector may furthercomprise, in addition to transfection or transformation, cellularaction, e.g. in the case of RNA virus-based precursor vectors, a primarytransformed or transfected DNA may require transcription in order toproduce the RNA precursor vector in the cell. In the case ofAgrobacterium-mediated delivery, the precursor vector may have to beexcised or transcribed from T-DNA delivered by Agrobacterium.

A replicon is a DNA or RNA molecule capable of autonomous replication ina cell of said plant. Examples include: bacterial and yeast plasmids,plastids and mitochondrial DNA, DNA and RNA viruses, viroids, phages. Areplicon has to have an origin of replication. The origin of replicationmay be recognized by a DNA or RNA polymerase of the host plant cell,depending on whether the replicon is a DNA or an RNA replicon, or by aheterologous polymerase e.g. of viral origin. In this case, the plantcell has to be provided with said heterologous polymerase e.g. by one ofsaid replicons. The autonomous replication of the replicons in the plantcell has presumably the effect that their concentration is increased,which may increase the expression level of the sequences of interest andsupport spread from cell-to-cell and throughout the plant. Preferably,replicons have an increased infectivity and ability to spread comparedto precursor vectors.

Said at least one or said at least two replicons may retain additionalviral capabilities such as viral particle assembly, infectivity,suppression of gene silencing, reverse transcription, integration intothe host chromosome, cell to cell movement, or long distance movement.One of said replicons may essentially be a helper type virus whichprovides in trans functions necessary for replication of anotherreplicon or replicons. Further, one of said replicons may essentially bea wild-type retrovirus or retrotransposon which provides in transfunctions necessary for replication, reverse transcription, integrationinto a host chromosome of another replicon or replicons.

Said at least one or said at least two replicons provide, preferablytogether, for the amplification and/or expression of said nucleic acidsequences of interest. If one sequence of interest is amplified orexpressed from one of said replicons another replicon may be requirede.g. for a function necessary for the replication of said replicons orfor spreading of at least one replicon to neighboring cells if saidprocess is performed in cell culture or in a plant. In this case, thereplicons cooperate functionally with each other.

If more than one sequence of interest is to be amplified or expressed,each sequence may preferably be expressed from one replicon, whereby thereplicons provide (together) for said amplification or expression. Alsoin this case, the replicons preferably cooperate functionally with eachother. As an example, a function for replication or spreading of saidreplicons may be expressed from one or some of said replicons whichamplify or express said sequences of interest or from an additionalreplicon. Without functional cooperation, the amplification orexpression level would be much lower or be limited to the cell(s)provided with said precursor vector(s), if amplification or expressionis desired in plant cells or a plant.

Said at least one or said at least two replicons are functionallydistinct from each other in that they provide different functions foramplification and/or expression of said sequence(s) of interest.Examples for such functions include, in addition to coding of saidnucleic acid sequence(s) of interest, the expression of productsnecessary for replicating the replicons, expression of factors orproducts (preferably enzymes) which facilitate the processing of theprecursor vector(s) to give said replicons, expression of productsnecessary for cell to cell or long distance or plant to plant movementof said replicons (e.g. movement protein or coat protein) etc. Theseproducts may function in trans or in cis. Functional distinctness doesnot include random mutations in the replicons which may occur in thecourse of the processing in the plant cell.

Owing to said processing, said at least replicons are structurallyrelated to each other in that they share sequence portions with eachother. The type of the relatedness depends on the type of processing(modification process). If one replicon is produced in said process,said replicon is structurally related to said at least one or to each ofsaid at least two precursor vectors owing to said processing.

The precursor vectors undergo processing in the plant cell by one of thefollowing DNA or RNA modification processes, which endows the plant cellwith said replicon or replicons. The processing in the plant cell mayinvolve DNA modification such as DNA recombination, insertion orexcision etc. Alternatively, it may involve RNA modification such as RNAsplicing, ligation or recombination etc. Said processing does notinclude transcription. The precursor vector may itself be a DNA or RNAmolecule capable of autonomous replication in a cell of said plant.

The precursor vector(s) are preferably of plant viral origin, morepreferably of an RNA virus or DNA virus origin. Examples of specificviruses are given below. Precursor vectors may be capable of autonomousreplication in the plant cell as are replicons.

The plant cell may be provided with one or more precursor vectors. Ifthe cell is provided with only one precursor vector, this precursorendows the plant cell with at least two replicons. If the cell isprovided with two or more precursor vectors, the cell is preferablyendowed with said at least one or said at least two replicons by aprocessing which involves interaction between said precursor vectors,e.g. recombination. Providing the plant cell with two or more precursorvectors greatly increases the possibilities for generating said repliconor replicons. Several different DNA or RNA modifications may occur inthe plant cell depending on the design of the precursor vectors.

This process is based on the use of a wild-type plant cell(s) or on aplant cell(s) that is (are) genetically engineered so as to providefunctions necessary for said processing, or to provide in trans one ormore functions necessary for infectivity, replicon replication, virusparticle assembly, suppression of gene silencing by the host,integration into a host chromosome, reverse transcription, cell to cellor long distance movement of said resultant replicons. Said geneticengineering of said plant cell is done by transient expression, virus-or Agrobacterium-mediated transfection, stable integration into genomesof nuclei or organelles or into autonomously replicating plasmids ofsaid plant cell. The plant cells and plants for the process describedhere are preferably higher plants or cells thereof. Crop plants areparticularly preferred.

The process of causing amplification or expression features severalimportant advantages over the prior art. The size of the nucleic acidsequence(s) of interest to be amplified or expressed is far less limitedthan in the prior art, since functions necessary for amplification orexpression are shared by at least two replicons, whereby the repliconsare smaller than a prior art vector would be. Consequently,amplification or expression is more efficient.

Furthermore, this process allows the amplification and/or expression ofmore than one nucleic acid sequence of interest. Examples are providedfor the expression of two genes of interest. However, the expression ofthree, four or even more nucleic acid sequence of interest is alsofeasible. This allows the expression of a whole biochemical pathway orcascade or of a multi-subunit protein. Each sequence of interest maypreferably be expressed from one replicon. Additional function(s) forefficient performance of the process like those listed below may belocated on an additional replicon. Alternatively, a replicon may encodemore than one of these functions.

A third important advantage is that there are several possibilities ofimproving the biological safety over prior art processes. In embodimentswherein more than one precursor vector is employed, the process is onlyfunctional when all precursors get into the plant cell. Alternatively,the processing in the cell to generate said at least one or said atleast two replicons may be dependent on an additional component e.g. anenzyme which catalyses said processing. Then the system is onlyfunctional if the additional component is delivered into the cell or ifa transgenic cell expressing said component is used. In one embodiment,the replicon which expresses a sequence of interest can spreadthroughout the plant from the primary infected cell, but spreading toother plants is not possible.

Examples for nucleic acid sequences of interest include coding sequencesor parts thereof, or any genetic element. Herein, a genetic element isany DNA or RNA element that has a distinct genetic function other thancoding for a structural part of a gene. Examples include:transcriptional enhancer, promoters, translational enhancers,recombination sites, transcriptional termination sequences, internalribosome entry sites (IRESes), restriction sites. A preferred geneticelement is a tobamoviral IRES_(mp)75 used as translational enhanceroperably linked to a heterologous nucleic acid sequence encoding aprotein of interst.

In a preferred embodiment, said plant cell is endowed with at least one(type of) replicon. Said one replicon is preferably formed bysite-specific recombination between at least two precursor vectors. Saidsite-specific recombination preferably takes place on DNA level. Saidone replicon may therefore be DNA. Alternatively, said one replicon maybe RNA formed by transcription following said site-specificrecombination. In this embodiment, said precursor vectors are preferablynot capable of autonomous replication in said plant cell. Thisembodiment is particularly preferred from the point of view ofbiological safety.

In another preferred embodiment, a plant cell is provided with the cDNAof a precursor vector (FIGS. 17 and 19). After transcription whichproduces the precursor vector, processing in the cell by splicing endowsthe cell with an RNA virus-based replicon from which a sequence ofinterest can be amplified or expressed. Since splicing is slow and/ordoes not happen in all the precursor vector molecules in the cell,unspliced precursor molecules will remain in the cell. These remainingunspliced precursor vectors are replicons as well provided they arecapable of autonomous replication. They are used for the expression of(a) function(s) necessary for amplification or expression of saidsequence of interest, e.g. a function for spreading of the replicon(s)to neighbor cells.

In another preferred embodiment, a set of replicons of the type AB₁,AB₂, . . . , AB_(n) or of the type B₁A, B₂A, . . . , B_(n)A aregenerated in a cell by site-specific recombination of a primaryprecursor vector (A) with a set of at least two secondary precursorvectors (B₁, B₂, . . . , B_(n)) wherein n is an integer of ≧2. In theembodiment shown in FIG. 23, precursor vectors (secondary vectors)containing a sequence to be expressed or amplified are recombined withanother precursor vector (primary precursor vector) to form replicons ofthe type AB₁, AB₂ and AB₃, from which these sequences can be amplifiedor expressed. At least three precursor vectors are needed to endow thecell with at least two replicons. Preferably, functions for spreadingthe replicons are expressed from one or more of said replicons.

For performing the above processes, precursor vectors may directly beused for transforming or transfecting a plant or plant cell. This isparticularly true for DNA virus-based replicons. For RNA virus-basedreplicons, DNA vectors used for transformation or transfection requiretranscription for producing the precursor vectors inside the cell.

The processes described here may be used for generating high amounts ofone or more nucleic acid sequences in a plant, plant tissue, plant cellor cell culture in an environmentally safe way. Notably, said processesmay be used for generating high amounts of said at least one or said atleast two replicons. Said replicon(s) may be purified or enriched fromthe plant material. Said replicon(s) may be vectors or viral materialthat may be used, optionally after enrichment or purification, fortransforming or transfecting a further plant, plant tissue, plant cellsor cell culture. In this embodiment, the subject processes may bebiologically safe processes of producing infectious viral material orvectors for transforming or transfecting plants on a large scale like onan agricultural or farming scale. Said infectious viral material or saidvectors may be packaged or coated viral material. The protein materialnecessary for packaging said viral material may be expressed from saidreplicon(s).

Detailed Description of Pro-Vector System

Viruses belonging to different taxonomic groups can be used for theconstruction of virus-based vectors according to the principles of thepro-vector system. This is true for both RNA- and DNA-containingviruses, examples for which are given in PCT/EP02/03476.

Mostly, vectors of plant viral origin are used as plasmids capable ofautonomous replication in plants (replicons). However the principlesnecessary for engineering such plasmids using non-viral elements areknown. For example, many putative origins of replication from plantcells have been described (Berlani et al., 1988, Plant Mol. Biol., 11,161-162; Hernandes et al., 1988, Plant Mol. Biol., 10, 413-422; Berlaniet al., 1988, Plant Mol. Biol., 11, 173-182; Eckdahl et al., 1989, PlantMol. Biol., 12, 507-516). It has been shown that the autonomouslyreplicating sequences (ARS elements) from genomes of higher plants havestructural and sequence features in common with ARS elements from yeastand higher animals (Eckdahl et al., 1989, Plant Mol. Biol., 12,507-516). Plant ARS elements are capable of conferring autonomousreplicating ability to plasmids in Saccharomyces cerevisiae. Studies ofmaize nuclear DNA sequences capable of promoting autonomous replicationof plasmids in yeast showed that they represent two families of highlyrepeated sequences within the maize genome. Those sequences have acharacteristic genomic hybridization pattern. Typically there was onlyone copy of an ARS-homologous sequence on each 12-15 kb of genomicfragment (Berlani et al., 1988, Plant Mol. Biol., 11:161-162). Anothersource of replicons of plant origin are plant ribosomal DNA spacerelements that can stimulate the amplification and expression ofheterologous genes in plants (Borisjuk et al., 2000, Nature Biotech.,18, 1303-1306).

Therefore, a replicon or precursor vector contemplated here is notnecessarily derived from a plant virus. Plant DNA viruses provide aneasy way of engineering replicons (vectors) that could be especiallyuseful for targeted DNA transformation, but vectors made entirely orpartially of elements from plant RNA viruses or even non-plant virusesare possible. Plant virus-based replicons are evidently advantageous.Such replicons, in addition to replication, may provide additionaluseful functions e.g. for cell to cell and long distance movement.Further, they can frequently be removed more easily from the plant cellaposteriori by using known methods of virus eradication from infectedplants.

In the simplest case, one type of precursor vector (pro-vector) (A) isdelivered into the plant cell where, upon its processing in said cell,it yields at least one or at least two structurally related andfunctionally distinct replicons (F and G) which are able to provideamplification and/or expression of the sequences of interest. More thanone precursor vector can be introduced into the plant cell [A(+C,D,E . .. )]. Optionally, said plant cell can be transgenic and contain anotheradditional component (B) required for the processing of the precursorvector and/or expression, replication, cell-to-cell or systemicmovement.

In one embodiment, we describe a pro-vector (precursor vector) systemthat is based on the splicing of the pro-vector RNA in the plantnucleus. This system comprises a DNA molecule of the pro-vectorcontaining parts of plant virus genome cDNA, gene(s) of interest andsplicing-sites functional in the plant cell (see for example FIG. 17 or19). After transcription in the transfected plant cell, the primary RNAproduct (pro-vector or precursor vector) can be processed by splicing.Due to the design of the pro-vector (precursor vector), the gene ofinterest (e.g. GFP) substitutes one of the viral genes (e.g. CP) in thisspliced form. This allows the gene of interest (GFP) to be expressed viathe normal viral pathway instead of substituted viral protein. However,since splicing cannot proceed with 100% efficiency, a fraction of theprimary transcript remains unspliced and is exported from the nucleus asis the spliced form. As a result, two types of self-replicating viralvector RNAs (replicons) appear in the cytoplasm of the transfected plantcell. This leads to the expression of both GFP and CP from repliconsgenerated using one precursor vector. It must be mentioned that, in theexemplified case, the level of GFP expression is likely much higher thanthat of CP. It was shown for tobamoviruses that the amount of viralprotein produced during infection depends on the distance between geneencoding the protein and 3′-terminus of the virus. Since GFP isexpressed from the spliced form of RNA, the corresponding subgenomic RNAis significantly shorter than the RNA from which CP is expressed (FIG.19; sGFP stands for a synthetic or engineered GFP).

As an exemplification of this embodiment we constructed two pro-vectors(precursor vectors) based on two well-known plant viruses: potato virusX (PVX) and crucifer infecting tobamovirus (CrTMV). In both cases, theCP gene of the viruses was flanked with donor (upstream) and acceptor(downstream) splicing sites (SA and SD, respectively). GFP was cloneddownstream of the acceptor site to be expressed only in the splicedtranscript. The physiological roles of CP in PVX and CrTMV aredifferent. In the case of PVX, CP is required for cell-to-cell movementof the virus. In CrTMV, CP participates mainly in long-distance spreadof the virus (systemic infection) and is not crucial for infection ofneighbor cells. These examples provide two different patterns ofreporter gene (GFP) expression. In the case of the PVX-based system,viral spread is stalled in primary transfected cells until the requiredamount of CP is expressed from a less efficient replicon that was formedfrom unspliced RNA. This leads to super-production of much more rapidlysynthesized GFP in the same cell. Finally, the necessary amount of CPaccumulates and both replicons penetrate neighbor cells where theprocess repeats. On the contrary, in the case of the CrTMV-basedpro-vector, viral spread is not limited to cell-to-cell movement. Bothforms of the vector act independently, which leads to faster growth ofthe infected area.

Although specific examples describing RNA modification as a mechanismfor generating replicons are based on RNA splicing, other RNAmodification mechanisms may be used as well. These include inter aliamodifications as RNA ligation or RNA recombination. Ligation ofdifferent RNA molecules by enzymes such as RNA ligase allows to producea plurality of different RNA replicons within a cell based on theinternal enzymatic activity of plant cells or based on the expression ofwell known ligases such as T4 RNA ligase (Nishigaki et al., 1998, Mol.Divers., 4, 187-190) or mitochondrial RNA ligase from Leishmania (Blancet al., 1999, J. Biol. Chem., 274, 24289-24296). RNA-RNA recombinationis a well researched phenomenon. It readily occurs in a plant hostbetween viral RNA molecules with a certain degree of homology (Allisonet al., 1990, Proc. Natl. Acad. Sci. 87, 1820-1824; Rao et al., 1990, J.Gen. Virol., 71, 1403-1407; U.S. Pat. No. 5,877,401).

In another embodiment, the precursor vectors are processed bysite-specific DNA recombination producing a replicon or partiallydifferent replicons or assembling one viral replicon in vivo. In thiscase, molecular rearrangements proceed on the DNA level. Severalmolecules (pro-vectors) are shuffled by means of recombination toproduce one or several vector molecules (replicons). The firstDNA-component of the system may contain a plant promoter (ArabidopsisActin 2) fused to the main part of CrTMV genome—the polymerase gene plusthe MP gene followed by a specific recombination site. Secondarycomponents are plasmid DNAs comprising the same recombination sitefollowed by a gene(s) of interest (any reporter gene) or viral CP gene.The 3′-UTR of CrTMV should be cloned downstream of the gene of interest.The last component of the system may be Cre-recombinase or integraseexpressing DNA. These enzymes promote reorganization of pro-vectorcomponents into active vector molecules (replicons). It must be stressedthat none of the pro-vector components is infectious alone, which isimportant in terms of the biological safety of the system.

After providing a plant cell with all the components of the describedmulti-component system, recombination occurs and one or severalreplicons can be formed (see FIGS. 23 A,B,C). These rearranged DNAmolecules can be transcribed (since they carry Arabidopsis Actin 2promoter) in the nucleus and exported to the cytoplasm. Finally, like inthe case involving RNA splicing, several replicating vector RNAs appearin the cytoplasm of the transfected cell. In this embodiment, wedescribe the system where all of these vectors contain a functional MPgene and a gene positioned downstream. This allows each vector RNA toexpress both a functional MP gene and a gene positioned downstream andto penetrate into neighbor cells. One has to state that othercombinations are also possible.

Two different approaches of recombinase delivery are exemplified. Therecombinase may be delivered into the cell in a process ofco-bombardment or by Agrobacterium-mediated transient expressiontogether with other DNA-components of the system. As another approach, atransgenic plant expressing cre-recombinase has been obtained. Thisreduces the number of components the cell has to be provided with and asa result raises the overall efficiency of the system. Additionally, thisfurther improves the safety of the process as it cannot occur outside ofa plant that was genetically manipulated to support the process.

During the cloning of pro-vector components, a LoxP recombination sitewas cloned upstream of the gene(s) of interest. A LoxP site contains twosmall inverted repeats spaced by several nucleotides and it formsstem-and-loop structure in RNA. The stem contains only 4 GC pairs so itis not very stable. However, this stem can reduce the efficiency oftranslation of a downstream gene. To solve this problem, anytranslational enhancer can be cloned between LoxP and the gene ofinterest. In the examples with Cre recombinase we used an omega leaderof TMV U1. However any other translation-enhancing sequence can be used(e.g. IRES_(mp)75 from CrTMV or TMV U1) as well as plant IRESes.IRES_(mp)75 elements may preferably be used as translational enhancersas in the examples with integrase of phage PhiC31. In this case, in aset of provectors the LoxP recombination sites were replaced byatt-sites (attachment-sites) from the Streptomyces phage PhiC31 (Thorpe& Smith, 1998, Proc. Natl. Acad. Sci., 95, 5505-5510; Groth et al.,2000, Proc. Natl. Acad. Sci., 97, 5995-6000) which are the targetsequences for the recombination enzyme integrase. Two differentatt-sites were cloned into the provectors: Whereas attP was inserted inthe vectors of the type polymerase-MP-LoxP, an attB-recombination sitewas cloned into the provectors, which carry the gene of interestfollowed by a 3′NTR sequence and a nos-terminator. Similar to the Cresystem, the attB-recombination-sites limit the efficiency of translationof a gene, which is located downstream. Hence, also in this systemtranslational enhancer sequences were cloned between the attB-sites andthe genes of interest. It was shown that IRES_(mp)75 sequences havecomparable or even better effect as translational enhancers incomparison with the omega leader of TMV U1.

Suitable recombinases/recombination site systems include inter alia theCre-Lox system from bacteriophage P1 (Austin et al., 1981, Cell, 25,729-736), the Flp-Frt system from Saccharomyces cerevisiae (Broach etal., 1982, Cell, 29, 227-234), the R-Rs system from Zygosaccharomycesrouxii (Araki et al., 1985, J. Mol. Biol., 182, 191-203), the integrasefrom the Streptomyces phage PhiC31 (Thorpe & Smith, 1998, Proc. Natl.Acad. Sci., 95, 5505-5510; Groth et al., 2000, Proc. Natl. Acad. Sci.,97, 5995-6000), and resolvases. In addition, other methods of DNArearrangement are contemplated. Other DNA modification enzyme systemscan all be used to generate related but functionally distinct repliconsinside of a wild-type or a genetically engineered plant cell:restriction endonuclease, transposase, general or specific recombinase,etc.

Different methods may be used for providing a plant cell with precursorvectors. DNA may be transformed into plant cells by a Ti-plasmid vectorcarried by Agrobacterium (U.S. Pat. No. 5,591,616; U.S. Pat. No.4,940,838; U.S. Pat. No. 5,464,763) or particle or microprojectilebombardment (US 05100792; EP 00444882B1; EP 00434616B1). Other planttransformation methods can also be used like microinjection (WO09209696; WO 09400583A1; EP 175966B1), electroporation (EP00564595B1;EP00290395B1; WO 08706614A1) or PEG-mediated transformation ofprotoplasts etc. The choice of the transformation method depends on theplant species to be transformed. For example, microprojectilebombardment is generally preferred for monocot transformation, while fordicots, Agrobacterium-mediated transformation gives better results ingeneral. Agrobacterium-mediated delivery of precursor vectors ispreferred.

Construction of plant viral vectors for the expression of non-viralgenes in plants has been described in several papers (Dawson et al.,1989, Virology, 172, 285-293; Brisson et al., 1986, Methods inEnzymology, 118, 659; MacFarlane & Popovich, 2000, Virology, 67, 29-35;Gopinath et al., 2000, Virology, 267, 159-173; Voinnet et al., 1999,Proc. Natl. Acad. Sci. USA, 96, 14147-14152) and can be easily performedby those skilled in the art.

The current processes have a number of advantages compared to existingviral vector-based strategies to express foreign genes in plants.

Most importantly, the process can be used for the expression of morethan one nucleic acid sequence of interest and can thus be used for theexpression of multiple genes of a biochemical pathway or cascade. Inthis regard, the processes described here are the only available methodthat can be effectively used for the expression of genes for the purposeof gene function determination or for the purpose of biochemicalproduction.

The invention also opens up a wide range of opportunities for any kindof biological cascade construction. One example is presented in FIG. 28.This system utilizes recombination of 3 precursor vector components into2 replicons. First, replicon A expresses MP and a gene of interest (GFPin the example). Due to the expression of the movement protein (MP),this vector is able to move from cell-to-cell in the inoculated leaf.However it cannot spread systemically in the infected plant and cannotbe transmitted to other plants due to the absence of the coat protein(CP). In other words, this replicon is infectious only if it isartificially delivered into a plant cell. The second replicon Bexpresses only CP. Note that it is not able to form viral particlessince its RNA lacks the origin of virus assembly (positioned inside theMP gene). However it does express CP in significant amounts.

The proposed process is inherently more safe as it is operable only inthe presence of all the precursor vectors. If both of the repliconsdescribed above are present in the same cell, they complement each otherand both components are able to move to neighbor cells. However onlyvector A can be coated with CP to form viral particles and thereforeonly this component will be exported from the infected leaf into thewhole plant. If such viral particles penetrate uninfected leaves, theydeliver only the infectious component A, but not B. This leads to thesystemic spread of infection in the whole plant but the virus cannotinfect other plants because viral particles can be formed only inprimary inoculated leaves and these particles contain only one repliconcomponent, which is not enough for systemic infection of another plant.This system represents a unique example of highly efficient expressionof a transgene in the whole plant via a noncontagious viral vector.

Additionally, the process of assembly of one viral replicon from atleast two replicon precursors through site-specific DNA recombinationguaranties a higher level of safety in comparison with convenient viralvector being used for infecting plant cells. Said process of viralreplicon assembly from at least two components requires the presence ofsaid two components and a site-specific recombinase to be present in thesame plant cell. Said recombinase can be delivered in cis together withone of said components. Preferably, the recombinase can be deliveredseparately. More preferably, the host plant can be engineered toexpresses said recombinase. In the latter case, assembly of functionalvector from provector elements will be restricted specifically toengineered host plant.

The pro-vector system is exemplified in reference examples 4 to 17.

EXAMPLES Example 1

Construction of Vectors Carrying Different Types of GFP Fusions with theTobacco Pectin Methylesterase (PME) Gene for Transient Expression andStable Nuclear Transformation of Plants

A red-shifted mutant of GFP (GFPst65T) was chosen for fusion with PME todetermine the location of GFP in vivo. The following four constructswere created: 1) full length PME-GFP; 2) GFP-mature PME; 3) GFP-fulllength PME and 4) mature PME-GFP. The first construct was designed fortargeting the protein of interest (GFP) to the cell wall. Constructs 24were used as negative controls.

Plasmid pUC8 containing a full sequence of an unprocessed PME gene withuntranslated 5′ and 3′-regions was used for PCR amplification with thefollowing primers:

-   a) 5′-TGACCCATGGTGGATTCCGGCMGAACGTT-3′, corresponding to the    N-terminal part of the PME coding sequence and containing a NcoI    site.-   b) 5′-TTttGGATCCACGGAGACCAAGAGAAAAAG-3′, corresponding the    C-terminal part of the PME coding sequence and containing a BamHI    site. This pair of primers was designed for generating PME without a    translation termination signal.-   c) 5′-TGACGGATCCTTGGATTCCGGCMGMCGTTAAT-3′, corresponding to the    N-terminal part of the coding sequence of full length PME and    containing a BamHI site.-   d) 5′-CCCCTCTAGATCAGAGACCMGAGAAAAAGGGM-3′, corresponding to the    C-terminal part of full length PME and containing a XbaI site.

This pair of primers was designed for creating PME without the firstmethionine residue but with a translation termination signal.

-   e) 5′-TTTTCCATGGTGAGGCCCGACGTGGTTGTGGC-3′ corresponding to the    N-terminal part of the mature PME gene containing a translation    start codon in the NcoI site. This primer was designed for creating    a mature PME gene with a translation start but without a translation    termination signal.-   f) 5′-ATAAGGATCCGTGAGGCCCGACGTGGTTGTGGC-3′, corresponding to the    N-terminal part of the mature PME gene without translation    termination signal within the BamHI site. This primer was designed    for creating mature PME gene without start codon but with a    translation termination signal.

Different types of PCR products were cloned into pGEM-T vector (Promega)under the control of the T7 promoter, resulting in plasmids plC2406{primers a) and b)}; plC2396 {primers b) and c)}; plC2425 {primers a)and f)} and plC2415 {primers b) and f)}.

Then the GFP gene was modified for N- or C-terminal translational fusionwith PME. Two modifications of the GFP gene were cloned into the pGEM-Tvector under the control of the T7 promoter. GFP with a translationstart codon and without a translation termination signal was obtainedusing primers g) and h) (plC2441). GFP without a translation start codonbut with a translation termination signal was obtained using primers l)and j) (plC2431).

Used GFP Primers:

-   g) 5′-TTTTCCATGGTGAGCAAGGGCGAGGAGCTG-3′ corresponding to the    N-terminal part of the GFP gene and containing a translation start    codon within the NcoI site.-   h) 5′-TTTTGGATCCTATTCTTGCGGGCCCTCGCTTGTACAGCTCGTCCATGCC-3′    corresponding to the C-terminal end of the GFP gene and containing    BamHI restriction site.-   i) 5′-TTTTGGATCCATAAGMCGGGGCCCAGAGTGAGCMGGGCGAGGAGCTGT-3′,    corresponding to the N-terminal part of the GFP gene and containing    a BamHI site.-   j) 5′-TTTTTCTAGATTACTTGTACAGCTCGTCCA-3′, corresponding to the    C-terminal part of the GFP gene and containing a translation    termination signal within XbaI site.

For PME-GFP fusions, the NcoI/BamHI fragment of plC2431 containing GFPwithout translation start codon was ligated with the large NcoI/BamHIfragments of plC2406 and plC2425, yielding constructs plC2452 andplC2462, respectively. The general scheme of the cloning procedures inshown in FIG. 4A.

For GFP-PME fusions, the small NcoI/BamHI fragment of plC2441 wasligated with the large NcoI/BamH1 fragment of plC2396 or with the largeNco1/BamH1 fragment of plC2411 resulting in plasmids plC2472 andplC2482, respectively. The general scheme of the cloning procedures inshown in FIG. 4B.

In order to check the correctness of the PME-GFP fusions, the constructsplC2452, 2462, 2472, and 2482 were used for in vitro translation inrabbit reticulocyte lysate (RRL). The results presented in FIG. 5 showthe intactness of different PME-GFP fusion products.

For transient expression experiments, the large NcoI-Klenow/SalIfragments of plC 2452, 2462, 2472 and 2482 were ligated with theSmaI/SalI large fragment of plC056 expression cassete between the 35Spromoter and the nos terminator sequences, yielding constructs carryingp35S:full length (fl) PME-GFP, p35S:mature (m) PME-GFP, p35S:GFP-flPMEand p35S:GFP-mPME fusions. The cloning scheme is shown in FIG. 6.

EXAMPLE 2

Transient Expression of the PME-GFP Fusion in Nicotiana benthamianaLeaves

Plasmid DNA Preparation

Plasmids carrying p35S:flPME-GFP, p35S:mPME-GFP, p35S:GFP-flPME, andp35S:GFP-mPME fusions were transformed into E. coli strain DH10B, maxipreps were grown in LB medium and DNA was purified using the Qiagen kit.

Microproiectile Bombardment

Microprojectile bombardment was performed utilizing the BiolisticPDS-1000/He Particle Delivery System (Bio-Rad). The cells were bombardedat 900-1100 psi, at 15 mm distance from a macrocarrier launch point tothe stopping screen and 60 mm distance from the stopping screen to atarget tissue. The distance between the rupture disk and the launchpoint of the macrocarrier was 12 mm. The cells were bombarded after 4hours of osmotic pretreatment.

A DNA-gold coating according to the original Bio-Rad's protocol (Sanfordet al., 1993, In: Methods in Enzymology, ed. R. Wu, 217, 483-509) wasdone as follows: 25 μl of gold powder (0.6, 1.0 mm) in 50% glycerol (60mg/ml) was mixed with 5 μl of plasmid DNA at 0.2 μg/μl, 25 μl CaCl₂ (2.5M) and 10 μl of 0.1 M spermidine. The mixture was vortexed for 2 minfollowed by incubation for 30 min at room temperature, centrifugation(2000 rpm, 1 min), washing by 70% and 99.5% ethanol. Finally, the pelletwas resuspended in 30 μl of 99.5% ethanol (6 μl/shot). A new DNA-goldcoating procedure (PEG/Mg) was performed as follows: 25 μl of goldsuspension (60 mg/ml in 50% glycerol) was mixed with 5 μl of plasmid DNAin an Eppendorf tube and supplemented subsequently by 30 μl of 40% PEGin 1.0 M MgCl₂. The mixture was vortexed for 2 min and than incubatedfor 30 min at room temperature without mixing. After centrifugation(2000 rpm, 1 min) the pellet was washed twice with 1 ml of 70% ethanol,once by 1 ml of 99.5% ethanol and dispersed finally in 30 μl of 99.5%ethanol. Aliquots (6 μl) of DNA-gold suspension in ethanol were loadedonto macrocarrier disks and allowed to dry up for 5-10 min.

The leaves of Nicotiana benthamiana were bombarded by these goldparticles coated with different plasmid DNA. The results of theexperiments presented in FIG. 7 show that the flPME-GFP fusion productis targeted to the cell wall.

EXAMPLE 3

Agrobacterium-Mediated Transformation of Arabidopsis thaliana

Construct Design

The large Nhe1-EcOR1 fragments of plasmids p35S:flPME-GFP,p35S:mPME-GFP, p35S: GFP-flPME and p35S:GFP-mPME described in Example 1werte ligated with the large Xba1-EcOR1 fragment of binary vectorplCBV1. Resulting plasmids plCBV flPME-GFP, plCBV1 mPME-GFP, plCBV1GFP-flPME and plCBV1 GFP-mPME contained the p35S:flPME-GFP,p35S:mPME-GFP, p35S:GFP-flPME and p35S:GFP-mPME expression cassettes inT-DNA region with the BAR gene as selectable marker. The T-DNA region ofone construct, plCBV flPME-GFP is shown in FIG. 8.

In Planta Transformation of Arabidopsis thaliana

The plasmids (carbenicillin resistant) were immobilized intoAgrobacterium tumefaciens (strain GV 2260) by electroporation. Thebacterial cells were grown in 300 ml 2YT media with antibiotics,collected by centrifugation and resuspended in 5% sucrose to OD₆₀₀=0.8.

A. thaliana plants were grown until flowering. Then flowering bolts ofArabidopsis plants were dipped in Agrobacterium solution under vacuumapplied for a few seconds. Transformed plants were kept in a dark placefor 24 hours at high humidity and then transferred into the greenhouse.The seeds were collected 3-4 weeks later, sowed in soil and sprayed with100 mg/L phosphinothricin, 0.01% Silvet. The treatment was repeated 2-3times depending on the efficiency of selection and the frequency of lategermination events. Transgenic Arabidopsis plants were used for studyingthe GFP localization within plant tissues.

Selection for Transformants

Two to four days after the bombardment, the filter paper with cells wastransferred to the plate with CIM supplemented with the appropriateselection agent (10-15 μg/ml PPT). Every seven days, the material wastransferred to fresh selection media. The plates were kept in the darkand after approximately 6 weeks the plant material was transferred toPetri plates with Morphogenesis Inducing Medium (MIM) (see Appendix)supplemented with the appropriate selection agent (10-15 pg/ml PPT). Theplates were incubated at high light intensity, 16 hours day length.

EXAMPLE 4

Cell Wall Targeting of GFP Using a Translational Fusion with the NtTLRPGene

The binary vector-based translational fusion of GFP with the tobaccocell wall protein TLPR (Domingo et al., 1999, Plant J., 20, 563-570;Accession number Y19032) was performed in the same way as described forPME-GFP fusion, except for different primers.

-   a) 5′-TGACCCATGGGTTCTMGGCATTTCTGTTTCTTG-3′, corresponding to the    N-terminal part of the NtTLPR coding sequence and containing a NcoI    site.-   b) 5′-TTTTGGATCCTTGTGMGGCTGMCTTCAGTAAG-3′, corresponding to the    C-terminal part of the NtTLPR coding sequence and containing a BamHI    site. The cloning strategy was similar to that of flPME-GFP fusion    described in Examples 1 and 3. The final construct containing the    p35S:NtTLPR-GFP expression cassette within the T-DNA borders is    shown in FIG. 9.

In order to introduce a protein cleavage site which can be cut preciselyat the predetermined place in front of any amino acid residue, theArabidopsis ubiquitin gene (UBQ11, U.S. Pat. No. 5,773,705) wasintroduced between the NtTLPR and the GFP genes. The primers used forUBQ11 amplification were:

-   c) 5′-TTTTGGATCCAGATCTTCGTAAAGACTTTGACCG-3′, corresponding to the    N-terminal part of the UBQ11 coding sequence and containing a BamHI    site;-   d) 5′-TTTTGGATCCTTGTGMGGCTGMCTTCAGTMG-3′, corresponding to the    C-terminal part of the NtTLPR coding sequence and containing a BamHI    site.

The BamH1 treated and gel-purified PCR fragment of the IBQ11 gene wasligated with the BamH1-digested and alkaline phosphatase treated vectorplCBV1 NtTLPR-GFP. The orientation of cloned fragment was checked by PCRusing different combinations of UBQ1 and GFP primers. The T-DNA regionof the resulting plasmid is shown in FIG. 8. Transient expressionexperiments and stable transformation of plants were performed asdescribed in Examples 2 and 3.

EXAMPLE 5

Expression of NtTLPR-GFP Fusion from crTMV Vector

The Nco1-Sal1 fragment of NtTLPR-GFP fusion was cloned into a crTMVvector (Fig.

11) and used for transfection of Nicotiana benthamiana, Nicotianatabacum and Brassica plants. Details of crTMV-based vector constructionand reporter gene expression via an IRES element are given in referenceexamples 1 to 3 and in WO 02/29068.

EXAMPLE 6

Use of Ca²⁺-pectate Binding Site as Polypeptide Capable of Binding to aCell Wall Component

In this example, a Ca²⁺-pectate binding site of apoplastic isoperoxidasefrom zucchini (APRX) (Carpin et al., 2001, The Plant Cell, 13, 511-520)is used. It was shown that this site binds in vivo as well in vitro topectin chains via cross-links formed by Ca²⁺-ions. This site has a ofmotif of four clustered arginines that expose their positive charges atthe surface of the peroxidase. Ca²⁺-chelating agents like EGTA or EDTAare able to suppress the ionic interaction to the pectin chains becauseof their stronger affinity to Ca²⁺-ions. The peptide of 27 amino acidresidues with α-helical structure was fused to the C-terminus ofcalreticulin targeting signal-GFP gene to create expression vectorplCH8600-C (FIG. 14) and the 3′ component (plCP9666-C, FIG. 14) of theviral “pro-vector system” (PCT/EP02/03476). The peptide contains threearginines of the Ca²⁺-pectate binding site which are mainly responsiblefor the binding of the APRX-protein to pectin.

The GFP-protein was isolated from agroinoculated N. benthamiana plantsin three different ways: a) from crude extracts; b) from intercellularfluid (IF); c) from plant tissue squashed and washed in the presence ofCa²⁺-containing buffer.

In the case of a), the GFP-protein was bound to the cell wall fractionof crude extracts in the presence of Ca²⁺. After centrifugation, thesupernatant was removed and the bound GFP-protein could be dissolved inan EGTA-buffer. In the case of b), GFP was eluted from the apoplastusing EGTA-containing buffer. The same buffer was used to extract GFPfrom the squashed plant tissue. Further SDS-PAGE and Westernblot-analysis confirmed comparative yields and purification efficienciesfor all three different purification procedures.

a) Cloning of Protein Purification Expression Vectors plCH8600-C andplCH9666-C

The GFP-sequence was amplified by PCR from construct plCP5290(35Sprom-sGFP—NOS term) to fuse the Ca²⁺-pectate binding site in frameto the C-terminal end of the GFP-gene. The sequence of the forwardprimer (5′-TTTTCC ATG GTG AGC MG GGC GAG GAG CTGT-3′) introduced a NcoI-site at the 5′-end of the GFP-sequence whereas the reverse primer(5′-TTTT GGA TCC TAT TCT TGC GGG CCC TCG CTT GTA CAG CTC GTC CAT GCC-3′)included an additional 24 bp spacer after the last codon of theGFP-sequence followed by a BamH I-site. This PCR-product was subclonedinto construct plCP5290 as Nco I/BamH I-fragment generating constructplCH8155 (35Sprom-sGFP spacer wth 8 amino acid residues without stopcodon-NOS transcription termination signal). The Ca²⁺-pectate bidingsite was introduced into construct plCH8155 by an adapter using thefollowing oligos: 5′-cgggatccat tgttaaccgt cttggttctc gtgaaggaactttctttag acaatttcgt g-3′ and 5′-tgctctagat tacctaatgt ttcccatcttaatcatagaa acacgaaatt gtctaaagaa agttccttc-3′. Protruding ends of thisadaptor correspond to BamHI- and Xba I-digested sites, which were usedfor subcloning of the Ca²⁺-pectate binding domain into constructplCP8155. The final construct plCH8600 was Cla1-Nco1 digested and usedfor cloning the calreticulin signal peptide (CSP) as Cla1-Nco1 fragment(Borisjuk et al., 1999, Nat. Biotechnol., 17, 466-469). The GFP-fusioncassette was also subcloned into the 3′-provector plCP9422 (not shown)as Nco I/Xba I-fragment yielding the 3′-provector plCP9666. The plCP9666was Cla1-Nco1 digested, gel-purified and ligated with Cla1-Nco1 fragmentof calreticulin signal peptide. The final construct, plCP9666-C is shownin FIG. 14.

b) Expression of GFP-Fusion Protein in N. benthamina Leaves AfterAgroinfiltration of Construct plCH8600-C and plCP9666-C

Agroinfiltration of tobacco plants was performed according to a modifiedprotocol described by Yang and colleagues (Yang et al., 2000, Plant J.,22, 543-551). Agrobacterium tumefaciens strain GV3101 transformed withappropriate constructs was grown in LB-medium supplemented withRifampicin 50 mg/L, carbencillin 50 mg/l and 100 μM (micromolar)acetosyringone at 28° C. Agrobacterium cells of an overnight culture (5ml) were collected by centrifugation (10 min, 4500 g) and resuspended in10 mM MES (pH 5.5) buffer supplemented with 10 mM MgSO₄ and 100 μMacetosyringone. Bacterial suspension was adjusted to a final OD₆₀₀ of0.8. In case of delivery of several constructs, Agrobacteriumsuspensions of different constructs were mixed before infiltration.Agroinfiltration was conducted on near fully expanded leaves that werestill attached to the intact plant. The bacterial suspension wasinfiltrated with a 5 ml syringe. By infiltrating 100 μl of bacterialsuspension into each spot (typically 3-4 cm² in infiltrated area) 8 to16 spots separated by veins could be arranged on a single tobacco leaf.Plants were further grown under greenhouse conditions at 22° C. and 16 hlight.

Construct plCP9666-C was co-infiltrated with the corresponding 5′pro-vector plCH4851 and the integrase construct plCP1010 (see FIG. 15).As the result of integrase-mediated recombination between attP and attBsites, a viral vector expressing GFP at high level was assembled.Instead of plCP1010, any other vector capable of expressing the PhiC31recombinase may be used.

The purification of the GFP fusion from infiltrated plant material wasperformed by using three different approaches:

1) Purification by Binding to Cell Wall Matrix:

Leaf material expressing the GFP-fusion construct was ground with anextraction buffer, containing 2 mM CaCl₂, 20 mM HEPES pH 7.0, 0.1%Triton-x 100 and 5 mM β-Mercaptoethanol. Samples were centrifuged at9500 g for 5 min at 4° C. after incubation on ice for 60 min. Thesupernatant was removed carefully and the pellet was resuspended in 20mM HEPES pH 7.0 containing 2 mM EGTA and 0.1% Tween 20 to dissolve theGFP-fusion protein. The GFP-content was analysed in supernatants ofwash- and elution buffers by Western blot-analysis.

2) Purification by Elution from Apoplast:

Leaf material was infiltrated with 50 mM Tris-HCl, pH 7.5, 2 mM CaCl₂, 2mM EDTA. The intercellular fluid (IF) containing secreted proteins, butnot GFP, was removed by low-speed centrifugation (2000-3000 g). Theprocedure was repeated with the second buffer (50 mM Tris-HCl, pH 7.5, 2mM EDTA). The GFP, eluted by the second extraction buffer, was used forWestern blot analysis. Different versions of extractingapoplast-targeted proteins can be found in several publications (Fireket al., 1993, Plant Mol. Biol., 23, 861-870; Voss et al. 1995, MolecularBreeding, 1, 39-50; De Wilde et al., 1996, Plant Science, 114, 233-24;Kinai et al., 1995, Plant Cell, 7, 677-688; Liu et al., 1996, PlantScience, 121, 123-131). The procedure described here, works well forisolation of protein fusion with a Ca²⁺-pectate binding site.

3) Purification by Extraction from Squashed Plant Tissue

The plant tissue was squashed between the rollers of sap extractor(Erich Pollahne, Hannover, Germany) three or seven (in case of viralprovector system) days after agroinfiltration in presence of 2 mM CaCl₂,20 mM HEPES pH 7.0, 0.1% Triton-x 100 and 5 mM β-Mercaptoethanol. Theremaining plant tissue was rinsed once with the same buffer and fusionprotein was eluted using 20 mM HEPES pH 7.0 containing 2 mM EGTA and0.1% Tween 20. The GFP-content was analysed in supernatants of wash- andelution buffers by Western blot-analysis.

EXAMPLE 7

Use of Ca²⁺-Pectate Binding Site for Producing a Cytotoxic Protein

A synthetic gene encoding the EcORI endonuclease (genomic sequenceaccession No. J01675), flanked by NcoI (5′ end) and BamHI (3′ end)restriction sites and containing an inserted plant splicesome-recognisedintron after 721 bp of EcORI cDNA. The following intron sequence (shownin bold) together with flanking EcORI endonuclease sequences (initalics) was used: 5′-tatactcaag gttcgtaaag aacttttta ttttatcagtgtagtttgag cagttgtgac atattgtagt tctttaatac tcatgaaat gttttaattttaatatgg agtatag gagatggga-3′. The 940 bp NcoI-BamHI fragment ofsynthetic sequence was ligated with the large NcoI-XbaI fragment ofplCH9666-C (FIG. 14) and small BamHI-XbaI fragment containingCa²⁺-pectate binding site yielding plasmid plCH9666-RIA (FIG. 16).Religation of large ClaI-NcoI Klenow-treated fragment of plCH9666-RIAyielded plCH9666-RI, where the EcORI endonuclease lacksapoplast-targeting signal peptide (FIG. 16).

The agrodelivery of plCH9666-RIA or plCP9666-RI together with the 5′pro-vector plCH4851 and the integrase construct plCP1010 was performedas described in example 6. As the result of integrase-mediatedsite-specific recombination, an amplification vector was assembled thatproduced the EcORI enzyme either targeted into apoplast (forplCH9666-RIA) or with cytosolic location (for plCP9666-RI).

The cytotoxic effect of EcORI in case of cytosolic localisation of EcORI(construct plCP9666-RI) was clearly evident three days later, and fivedays after agroinfiltration the leaf tissue was badly damaged. On thecontrary, apoplast targeting of the enzyme allowed to preserveagroinfiltrated plant leaves for much longer period of time (up to twoweeks). The isolation of enzyme from plant tissue was performed asdescribed in example 6 (1. “Purification by binding to cell wallmatrix”) from plant material three and five days after agroinfiltration.The enzyme concentration in activity units was measured by adding 1 μLof different x10-x10⁴ dilutions to 20 μL of EcORI reaction buffercontaining pBS(KS+) with cloned 1.2 kb EcORI DNA fragment. Commerciallyavailable EcORI enzyme was used for determining the concentration. Thecomparative yield was approximately fifty times higher in case ofapoplast targeting of the enzyme.

REFERENCE EXAMPLES

The following reference examples 1 to 3 demonstrate the construction ofviral vectors and expression of the GUS reporter gene via an IRESelement. Further details can be found in WO 02/29068.

Reference Example 1

Construction of a tobamovirus Vector Infecting Cruciferous Plants

Virions of a known tobamovirus called crucifer tobamovirus (crTMV) whichis able to infect systemically crucifer plants were isolated fromOlearacia officinalis L. with mosaic symptoms. Results of crTMVhost-range examination are presented in Table1.

Plasmid Constructions

CrTMV cDNA was characterized by dideoxynucleotide sequencing (Dorokhovet al., 1994 FEBS Letters 350, 5-8). Full length T7 RNA polymerasepromoter-based infectious crTMV cDNA clones were obtained by RT-PCR fromcrTMV RNA using oligonucleotides crTMV1-Kpn5′-gcatggtaccccttaatacgactcactataGTTTTAGTTTTATTGCMCMCAACAA (upstream),wherein the italic bold letters are a sequence of a Kpn I site, theunderlined lowercase letters are nucleotide sequence of the T7 RNApolymerase promoter, the uppercase letters are from the 5′-termini ofcrTMV cDNA; and crTMV 2 5′-gcatgcggccgcTGGGCCCCTACCCGGGGTTAGGG(downstream), wherein the italic bold letters are sequence of NotI site,the uppercase letters are from 3′-termini of crTMV cDNA and cloning intopUC19 between KpnI and Bam HI restriction sites (FIG. 12).

Full length SP6 RNA polymerase promoter-based infectious crTMV cDNAclones were obtained by RT-PCR from crTMV RNA by using oligonucleotidescrTMV1-SP6 5′-gcatggtaccatttaggtgacactatagaactcG TTTTAGTTGCAACAACAACAA(upstream), wherein the italic bold letters are a sequence of a Kpn Isite, the underlined lowercase letters are a nucleotide sequence of theT7 RNA polymerase promoter, the uppercase letters are from the5′-termini of crTMV cDNA; and crTMV 25′-gcatgcggccgcTGGGCCCCTACCCGGGGTTAGGG (downstream), wherein the italicbold letters are a sequence of a Not I site, the uppercase letters arefrom 3′-termini of crTMV cDNA and cloning into pUC19 between KpnI andBam HI restriction sites (FIG. 12).

The full-length crTMV cDNA clones were characterized bydideoxynucleotide sequencing. The ability of crTMV infectioustranscripts to infect systemically Nicotiana and crucifer species wasconfirmed by infection tests on respectively Nicotiana tabacum var.Samsun and Arabidopsis thaliana.

Reference Example 2

Construction of Tobamoviral Vectors for Expression of GUS Genes inNicotiana and Crucifer Plants via Viral IRESs

Series of IRES-mediated expression vectors T7/crTMV/GUS were constructedas follows. First, Hind III and Xba I sites were inserted in the end ofthe CP gene of Sac II/Not I fragment of T7/crTMV vector (FIG. 12) by apolymerase chain reaction (PCR) and two pairs of specific primers.Second, IRES_(MP,75) ^(CR)-GUS, IRES_(MP,75) ^(UI)-GUS, IRES_(MP,228)^(CR) GUS, IRES_(CP,148) ^(CR)-GUS, IRES_(CP,148) ^(UI)-GUS, PL-GUS cDNAdescribed in Skulachev et al., (1999, Virology 263, 139-154) wereinserted into Hind III and Xba I containing Sac II/Not I fragment ofT7/crTMV vector to obtain Sac I-IRES_(MP,75) ^(CR)-GUS-Not 1, SacII-IRES_(MP,75) ^(UI)-GUS-Not I, Sac II-IRES_(MP,228) ^(CR)-GUS-Not I,Sac II-IRES_(MP,148) ^(CP)-GUS-Not I, Sac II-IRES_(MP,148) ^(UI)-GUS-NotI, Sac II-PL-GUS-Not I cDNA, respectively. Third, Sac II-Not I cDNAfragment of T7/crTMV vector was replaced by Sac I-IRES_(MP,75)^(CR)-GUS-Not I or Sac II-IRES_(MP,75) ^(UI)-GUS-Not I or SacII-IRES_(MP,228) ^(CR)-GUS-Not I or Sac II-RES_(CP,148) ^(CR)-GUS-Not Ior Sac II-IRES_(CP,148) ^(UI)-GUS-Not I or Sac II-PL-GUS-Not I cDNA toobtain respectively, vector T7/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 13),vector T7/crTMV/IRES_(MP,75) ^(UI)-GUS (FIG. 13), vectorT7/crTMV/IRES_(MP,228) ^(CR)-GUS (FIG. 13), vectorT7/crTMV/IRES_(CP,148) ^(CR)-GUS (FIG. 13), vectorT7/crTMV/IRES_(CP,148) ^(UI)-GUS (FIG. 13) and vector T7/crTMV/PL-GUS(FIG. 13).

Reference Example 3

Expression of GUS Gene in Transfected Nicotiana and Crucifer Plants viaViral IRESs

This example demonstrates the tobamovirus IRES-mediated expression ofthe GUS gene in Nicotiana benthamiana and Arabidopsis thaliana plantsinfected crTMV-based vectors: T7/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 13),vector T7/crTMV/IRES_(MP,75) ^(UI)-GUS (FIG. 13), vectorT7/crTMV/IRES_(MP,228) ^(CR)-GUS (FIG. 13, vector T7/crTMV/IRES_(CP,148)^(CR)-GUS (FIG. 13), vector T7/crTMWIRES_(CP,148) ^(UI)-GUS (FIG. 13)and vectorT7/crTMV/PL-GUS (FIG. 13).

In Vitro Transcription:

The plasmids T7/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 13), vectorT7/crTMV/IRES_(MP,75) ^(UI)-GUS (FIG. 13), vector T7/crTMV/IRES_(MP,228)^(CR)-GUS (FIG. 13), vector T7/crTMV/IRES_(CP,148) ^(CR)-GUS (FIG. 13),vector T7/crTMV/IRES_(CP,148) ^(UI)-GUS (FIG. 13) and vectorT7/crTMV/PL-GUS (FIG. 13) were linearized by Not I. The recombinantplasmids were transcribed in vitro as described by Dawson et al. (1986Proc. Natl. Acad. Sci. USA 83, 1832-1836). Agarose gel electrophoresisof RNA transcripts confirmed that they were intact. The RNAconcentration was quantified by agarose gel electrophoresis andspectrophotometry.

GUS Detection

Inoculated leaves were collected 10-14 days after transfection withcapped full-length transcripts. IRES activity was monitored byhistochemical detection of GUS expression as described earlier(Jefferson, 1987, Plant Molecular Biology Reporter 5, 387405). Sampleswere infiltrated using the calorimetric GUS substrate, but the method(De Block and Debrouwer, 1992, Plant J. 2, 261-266) was modified tolimit the diffusion of the intermediate products of the reaction: 0.115M phosphate buffer, pH 7.0 containing5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) 600 μg/ml; 3 mMpotassium ferricyanide; 10 mM EDTA. After incubation overnight at 37°C., the leaves were destained in 70% ethanol and examined by lightmicroscopy.

Reference Example 4

Construction of Spliceable PVX-Based Provector.

Two 35-PVX based provectors have been constructed. Plasmid PVX-201 wasused as a basic construct for cloning (Chapman S et al, 1992, Plant J1992 July;2(4):549-57). This plasmid contains the full-length cDNA ofthe potato virus X (PVX) genome (Gene Bank Accession Numbers NC 001455;AF172259) fused to the ³⁵S promoter and the nos terminator. The CPsubgenomic promoter region is duplicated and a few cloning sites areinserted between the duplicated regions in PVX-201 (see FIG. 18).

At the first step an intermediate construct plC2518 was obtained. Thisplasmid contains consequently: the C-terminus of CP (62 bp) from theXhoI site to the terminator with a HindIII site introduced right afterthe termination codon, enhanced GFP (sGFP) gene (starting codon includedinto NcoI site) and the 3′-terminus of PVX virus including theC-terminus of CP (62 nt), 3′-UTR and s poly (A) sequence followed by aSacI site (FIG. 18).

Two sets of oligonucleotides were synthesized to clone 2 different pairsof donor/acceptor splicing sites: D1+ AACGGTCGGTAACGGTCGGTAAA D1−TCGACTTTACCGACCGTTACCGACCGTT A1+ AGCTAACCTAGCAGGTTATATGCAGGTTATATGCAGGTCA1− TTGGATCGTCCAATATACGTCCAATATACGTCCAGGTAC 2. D2+ CGAAAGGTAAG D2−TCGACTTACCTTT A2+ AGCTAACCTATTGCAGGTTGC A2− CATGGCAACCTGCAATAGGTT

After annealing to each other, corresponding oligonucleotides form thefollowing double stranded DNA fragments: D1+/D1− fragment D15′ CGAACGGTCGGTAACGGTCGGTAAAG 3′ 3′ TTGCCAGCCATTGCCAGCCATTTCAGCT 5′A1+/A1− fragment A1 5′ AGCTAACCTAGCAGGTTATATGCAGGTTATATGC AGGTC 3′3′ TTGGATCGTCCAATATACGTCCAATATACGTCCA GGTAC 5′ D2+/D2− fragment D25′ CGAAAGGTAAG 3′ 3′ TTTCCATTCAGCT 5′ A2+/A2− fragment A25′ AGCTAACCTATTGCAGGTTGC 3′ 3′ TTGGATAACGTCCAACGGTAC 5′The 5′-protruding ends of the fragments are adhesive to ClaI/SalIrestricted DNA in case of D1 and D2. Fragments A1 and A2 can be ligatedinto HindIII-NcoI sites.

Plasmid PVX-201 described above (FIG. 18) was digested with ClaI andSalI. Then the D1 or D2 fragment was ligated into the digested vector,yielding construct plC3033 (in case of D1) of plC3053 (in case of D2).

Plasmid plC2518 (FIG. 18) was digested HindIII and NcoI. Ligation offragment A1 or A2 produced construct plC3041 (A1) or plC3068 (A2),respectively.

Two variants of a spliceable PVX provector plasmid were obtained bycloning of the XhoI-SacI fragment from plC3041 into plC3033 and of theXhoI-SacI fragment from plC3068 into plC3053. The resulting plasmidswere named plC3242 (splice sites D1+A1) and plC3258 (splice sites D2+A2)(FIG. 18)

REFERENCE EXAMPLE 5

Microprojectile Bombardment

Microprojectile bombardment was performed with the Biolistic PDS-1000/HeParticle Delivery System (Bio-Rad). Separate N. benthamiana leaves werebombarded at 900 psi with 15 mm distance from a macrocarrier launchpoint to the stopping screen and 60 mm distance from the stopping screento a target tissue. The distance between the rupture disk and a launchpoint of the macrocarrier was 12 mm. The cells were bombarded after 4hours of osmotic pretreatment.

The DNA-gold coating procedure (PEG/Mg) was performed as follows: 25 μlof gold suspension (60 mg/ml in 50% glycerol) was mixed with 10 μl ofplasmid DNA (up to 1 μg/μl) in an Eppendorf tube and supplementedsubsequently by 10 μl of 40% PEG in 1.0 M MgCl₂. The mixture wasvortexed for 2 min and than incubated for 30 min at room temperaturewithout mixing. After centrifugation (2000 rpm, 1 min), the pellet waswashed twice with 1 ml of 70% ethanol, once by 1 ml of 99.5% ethanol andfinally it was dispersed in 30 μl of 99.5% ethanol. Aliquots (6 μl) ofDNA-gold suspension in ethanol were loaded onto macrocarrier disks andallowed to dry up for 5-10 min.

Plasmid DNA Preparation

Plasmids were transformed into E. coli strains DH10B and JM109, maxipreps were grown in LB medium and DNA was purified using the Qiagen kit.

REFERENCE EXAMPLE 6

Mechanical Inoculation of Plants with Provector Plasmid DNA

Fully developed leaves of five to seven weeks old Nicotiana benthamianaplants were inoculated with plasmid DNA by mechanical wounding. For thispurpose, 10-50 μg of DNA was mixed with 3×GKP-buffer (50 mM glycine, 30mM K₂HPO₄,3% celite, 3% benthonite) and scratched gently on the upperside of the leaves.

REFERENCE EXAMPLE 7

Expression of a Reporter Gene in Plant Leaves by Converted (Spliced)PVX-Based Provector

Fully developed N. benthamiana leaves were bombarded with plasmidsplC3242 and plC3258. It was expected that two different RNA transcriptscan be synthesized in plant cell from each of these plasmids—completeform and spliced form (see FIG. 17).

The presence of the second transcript can be detected by the GFPfluorescence in a cell transfected with the provector.

Strong GFP fluorescence has been observed in numerous leave cellsbombarded with plC3242 (48 hours after bombardment). No GFP expressionwas detected in the case of plC3258—so this construct may serve as anegative control in this experiment. This difference occurred due to thedifferent donor/acceptor sites used in the constructs (see referenceexample 4). In the case of plC3258, a 9-nt sequences that representssplice-site consensus of Arabidopsis thaliana was used. In the case ofplC3242, donor and acceptor sites were designed as described in Nussaumeet al. Mol. gen. genet, 1995, 249:91-101 where they were tested andproved to be active in plants.

According to several investigations (Fedorkin et al., J Gen Virol 2001;82(Pt 2):449-58, Cruz et al., Plant Cell 1998; 10(4):495-510), the CP ofPVX is required for viral cell-to-cell transport. In case of theconstruct plC3242, the CP gene must be spliced out of RNA. This meansthat the spliced form of the transcript (FIG. 18) cannot express the CPand provide cell-to-cell movement of the virus. However, in severalexperiments with plC3242 not only single cells but also multi-cell loci(10-1000 cells) expressing GFP where detected. This indicates that thesplicing of the provector transcript does not occur with 100%efficiency. A fraction of the full length RNA remains unspliced andtherefore CP can be expressed from this form of the transcript, whilethe GFP gene remains silent in this RNA (since no GFP expression wasdetected in case of plC3258).

REFERENCE EXAMPLE 8

Generating Transgenic N. benthamiana Plants Expressing Cre Recombinase.

The actin2 promoter-LoxP-Cre Orf-Nos terminator fragment from plC1321was subcloned as a NotI blunt-SacI fragment into the SmaI and SacI sitesof the binary vector pBIN19, resulting in construct plC1593 (FIG. 29).

The plasmid plC1593 was introduced in Agrobacterium strain Agl1 byelectroporation and transformed agrobacteria were used to transform N.benthamiana. DNA extracted from 10 transformants was used to test forthe presence of the transgene by PCR. All plants were found positivewhen PCR was performed with primers for the kanamycin transformationmarker or for the Cre gene.

REFERENCE EXAMPLE 9

Generation of Functional Vectors from Provectors Using Site-SpecificRecombination

a) General Description of the System

The system described consists of two core types of CrTMV based vectorswhich carry LoxP-recombination sites (FIG. 23):

-   i) Vector type: Polymerase-MP-LoxP: The vector encodes the RdRP of    CrTMV and the movement protein (MP), which allows the virus to move    from cell to cell, but not to move systemically. The viral    transcription is controlled by an Arabidopsis-Actin 2 promoter.-   ii) Vector type: LoxP-gene of interest-3′NTR-nos-terminator (see    FIGS. 23 a-c): This class of vectors encode a reporter gene (sGFP or    GUS) and regulatory elements (nos: nopalin-synthase terminator; 3′    NTR: nontranslated region, pseudoknots and tRNA-like structure of    CrTMV). The expression of the coat protein (CP; see FIG. 23 b)    allows the vector to spread systemically in the host plant.-   iii) Vector type: Polymerase-MP-attP: See i), with    attP-recombination site-   iiii) Vector type: attB-gene of interest-3′NTR-nos-terminator (see    FIG. 30 a-c): See ii), with attB-recombination site

After recombination catalyzed by Cre-recombinase or integase (viacoinfection of a Cre or integrase encoding viral construct) functionalunits (replions) are formed which are able to express a reporter geneefficiently and are capable of moving from cell-to-cell (FIG. 23A-C, 30A-C) or systemically (FIG. 23B).

b) Plasmid Construction

A. Clonina of Vector Type Polymerase-MP-LoxP (FIG. 24)

An EcOR1-XhoI-fragment of MP was taken from plasmid plC3342 (FIG. 20)and was cloned into plasmid plC1212 which carries two LoxP-sites inopposite orientations. The MP gene of plasmid plC3342 contains aterminator codon which was introduced 25 M before the natural stop. Inthe resulting product plC3431, a fragment containing part of the MP geneis located next to a LoxP-site. Both elements are isolated viaEcORI-SacI restriction. After blunting the SacI restriction site, thefragment was cloned into the vector-containing part of plasmid plC3301,which contains a single EcORI restriction site in the MP gene. NotI(blunted) was chosen as a second restriction site. In the resultingligation product (plC3461) the CP-gene, the IRES-sequence, the gene forsGFP and the 3′ nontranslated region of plC3301 are replaced by LoxP(FIG. 24).

B. Cloning of LoxP-reportergene-3′NTR-nos-terminator vectors

a) Construct LoxP-sGFP-3′NTR-nos (FIG. 23 a)

The XhoI-NcoI-fragment which carries a LoxP-site next to anΩ-leader-sequence was taken from vector plC2744. In order to place thesequences adjacent to a reporter gene, the fragment was cloned intoplasmid plC1721, which contains the appropriate restriction sites nextto a gene for sGFP and a 3′NTR-sequence. Replacement of an IRES-sequenceby the fragment let to the resulting plasmid plC3421. In order to add anos-terminator-sequence, plasmid plC3421 was cut by KpnI and NotI. Thefragment was introduced into the vector-containing part of plasmid plC3232 and the final construct plC3441 could be obtained (see FIGS. 25 and23 a).

b) Construct LoxP-CP-sGFP-3′NTR-nos (FIG. 23 b)

Described above plasmids plC3342 and plC1212 were used as startingvectors. The PstI-SacI fragment of plC3342 that contains the genes forCP and sGFP was cloned into plC1212. As a result, a LoxP-site is locatednext to CP and sGFP in the product plC3451. In order to add anos-terminator sequence, a similar approach as in case a) was used: AnEcORI-NotI-fragment of plC3451 was introduced into the vector-containingpart of plC3232 resulting in plasmid plC3491 (see FIG. 26).

c) Construct LoxP-CP-GUS-3′NTR-nos (FIG. 23 c)

Plasmid plC751 is analogous to plC1721 and carries a GUS reporter geneinstead of sGFP. By cutting with NcoI and NotI the GUS-Gene and the3′-untranslated region can be directly cloned into plC3441 henceobtaining the final construct plC3521 (FIG. 27).

REFERENCE EXAMPLE 10

Expression of a Single Reporter Gene in Plant Leaves by Converted(Recombined) CrTMV-Based Provector

Separate N. benthamiana leaves were particle-bombarded with a mixture ofthree plasmids: plC3441 (LoxP-GFP, FIG. 25), plC3461 (Actin2promoter-CrTMV RdRp-MP-LoxP, FIG. 24) and plC2721 (Cre-Recombinase undercontrol of hbt promoter). GFP fluorescence was detected in severalmulti-cellular loci 38 hours after bombardment. A strong increase offluorescence and of the size of the infected area were observed duringthe following days (bombarded leaves where incubated at 25° C. on wetfilter paper). No GFP fluorescence was detected in control leavesbombarded with plC3441 together with plC2721 without plC3461.

REFERENCE EXAMPLE 11

Expression of Several Genes in Plant Leaves by Converted (Recombined)CrTMV-Based Provector

Separate N. benthamiana leaves were particle-bombarded with a mixture ofseveral plasmids:

-   a) plC3441 (LoxP-GFP, FIG. 25), plC3461 (Actin2 promoter-CrTMV    RdRp-MP-LoxP, FIG. 24) and plC2721 (Cre-Recombinase under control of    hbt promoter), plC3521 (LoxP-GUS, FIG. 27).-   b) plC3441 (LoxP-GFP, FIG. 25), plC3461 (Actin2 promoter-CrTMV    RdRp-MP-LoxP, FIG. 24) and plC2721 (Cre-Recombinase under control of    hbt promoter), plC3491 (LoxP-CP, FIG. 26).

Both reporter genes GFP and GUS were strongly expressed in bombardedleaves in case a). Viral particle formation and systemic movement of thevirus are take place in case b).

REFERENCE EXAMPLE 12

Construction of Spliceable CrTMV-Based Provector

Spliceable provector based on CrTMV virus has certain advantagescomparing to PVX-based one. Described in FIG. 19 system allows one tocreate reporter gene expressing system with the virus, which is fullyfunctional in the infected leaf. Contrary to PVX, coat protein of CrTMVis not required for viral cell-to-cell movement so splicing does noinfluence on the viral spread in the infected leaf.

A. Cloning of Intermediate Construct plC3312

A PCR fragment was obtained using cloned cDNA of CrTMV and the followingprimers:

-   MPERI+ (corresponds to middle-region of MP including EcORI    site—position 5455 in CrTMV genome): GTGGTTGACGAATTCGTC-   MP−(Complementary to C terminus of MP 17 aa upstream of the natural    termination codon introducing artificial terminator with downstream    ClaI and XhoI sites. Also this primer contains point mutation of CP    ATG into ACG which removes natural start of CP gene without amino    acid change in the MP):    GGTCTCGAGTTATCGATTATTCGGG-TTTGTMTGTTGTAAGACGTTTTCTTCTTTC

Another PCR fragment was obtained using the same template and thefollowing primers:

-   CP+(Corresponds to beginning of CP gene with XhoI and PstI sites    introduced upstream of ATG):    TMCTCGAGACCTGCAGCATGTCTTACAACATTACAAACC-CGMTCAG-   CP−(Complementary to C terminus of CP introducing single nucleotide    substitution to eliminate NcoI site in CP gene. Also this primer    introduce HindIII and NcoI restriction sites downstream of CP gene):    CTACTCCATGGTCMGCTTMGTAGC-AGCAGCAGTAGTCCACGGCACC

First, the PCR fragment was digested with EcORI and XhoI enzymes.Second, XhoI and NcoI digested fragments were ligated together intovector plC1721 (FIG. 20) by EcORI and NcoI sites yielding plasmidplC3151 (FIG. 20). Then plC3151 was digested KpnI-EcORV, the KpnI sitewas blunted with T4 DNA polymerase and the cut plasmid was selfligatedto eliminate the sites between KpnI and EcORV. The resulting plasmid wasnamed plC 3312 (see FIG. 20).

B. Cloning of Spliceable Provector plC3393 and Control ConstructionplC3401

The dsDNA fragments described in reference example 4 containing donorand acceptor splice sites were cloned into plC3312 using ClaI and XhoIsites in the case of donor and HindIII and NcoI in case of acceptorsplice sites (see FIG. 21). The obtained plasmids were named plC3378(donor site) and plC3382 (acceptor site). To obtain a negative controlconstruct, plC3401 EcORI-NotI fragment from plC3382 was cloned intoplC3301 (FIG. 22).

The final construct plC3393 was obtained in two-fragment cloning usingthe EcORI-PstI fragment from plC3378 with the PstI-NotI fragment fromplC3382 and plC3301 digested EcoRI and NotI as a vector (FIG. 22).

REFERENCE EXAMPLE 13

Expression of Reporter Gene in Plant Leaves by Converted CrTMV-BasedSpliceable Provector

Separate N. benthamiana leaves were particle-bombarded with plasmidsplC3393 and plC3401. GFP fluorescence was detected in severalmulti-cellular loci 48 hours after bombardment in case of plC3393. NoGFP fluorescence appeared in leaves bombarded with control constructplC3401.

REFERENCE EXAMPLE 14

Expression of One Gene of Interest from Viral Vector Assembled from TwoCrTMV-Based Provectors Through Site Specific att/integrase-BasedRecombination System

a) General Description of the System

The system described consists of two core types of CrTMV based vectorswhich carry attB- or attP-recombination sites (FIG. 30):

-   i) Vector type: Polymerase-MP-attP: The vector encodes the RdRp of    CrTMV and the movement protein (MP), which allows the virus cell to    cell, but not systemic movement. The viral transcription is    controlled by an Arabidopsis-Actin 2 promoter.-   ii) Vector type: attB-gene of interest-3′NTR-nos-terminator (see    FIG. 30 a-b): This class of vectors encodes a reporter gene (sGFP or    GUS) and regulatory elements (nos: nopalin-synthase terminator; 3′    NTR: nontranslated region, pseudoknots and tRNA-like structure of    CrTMV).-   iii) Vector type: attB-ubiquitin-gene of    interest-3′NTR-nos-terminator (see FIG. 30 c): In order to obtain a    defined protein which can be isolated from plants, an    ubiquitin-cleavage-signal-sequence was introduced between the    attB-recombination-site and the downstream located gene of interest    (e.g. GFP, Interferona2B, Insulin or Somatotropin). By using this    system, any amino acid except prolin can be chosen as the first    amino acid of the synthesized protein.

After recombination catalyzed by the integrase-enzyme (via coinfectionof a integrase-encoding viral construct), functional units (replicons)are formed which are able to express the gene of interest efficientlyand are capable of cell-to-cell movement (FIG. 23A-C).

b) Plasmid Construction

A) Cloning of Vector Type: polymerase-MP-attP (FIG. 31)

An KpnI-XhoI-fragment fragment was taken from plasmid plC3461 (FIG. 31).After blunting the XhoI-restriction-site, the fragment was cloned intothe binary vector plC3994 which carries two attB-sites in directorientations, left- and right-T-DNA-borders (LB and RB) and aKanamycin-expression-cassette as a plant transformation marker. Like theanalogous clone from the Cre/Lox-system, the resulting plasmid plCH4851carries an MP gene with a terminator codon which was introduced 25 AAbefore the natural stop.

B. Cloning of the Vector-Type: attB-Gene ofInterest-3′NTR-nos-terminator (FIGS. 27-35)

a) Construct attB-sGFP-3′NTR-nos

Primer Combination A)

-   attB Xho (+): ATCACTCGAGCTCGAAGCCGCGGTGCGGGT: Complementary to the    5′-part of attB and carrying an XhoI-restriction site at the    5′-terminus-   attB Ω(−): GGTMTTGTTGTAAAAATACGATGGGTGAAGGTGGAGTACG: The 3′-part of    the primer corresponds to the 3′-region of the attB-sequence, the    5′-part contains 20 nucleotides with complementary to the 5′-part of    the Ω-element.

Primer combination A was used on an attB-containing template in order tocreate a PCR-product which consists of the complete attB-sequence, 20 Ntof the 1-leader sequence and an XhoI-restriction-site at the 5′end.

Primer Combination B)

-   attB Ω(+): CGTACTCCACCTCACCCATCGTATTTTTACMCMTTACC:-    The 3′-part of the primer corresponds to the 5′-region of the    Ω-sequence, the 5′-part contains 20 nucleotides with complementary    to the 5′-part of the attB-element.-   Ω NcoI(−): CATGCCATGGGTAATTGTAAATAGTAATTGTMTGTT-    Complementary to the 3′-part of Ω and carrying an NcoI-restriction    site at the 5′-terminus

Primer combination B was used on an Ω-sequence-containing template inorder to create a PCR-product which contains the complete sequence ofthe Ω-leader, 20 Nt of the attB-recombination site and aNcoI-restriction-site at the 3′end.

After obtaining PCR products from primer combination A) and B), theisolated oligonucleotides were used together as templates for aPCR-reaction by using the primers attB XhoI (+) and Ω NcoI (−). As afinal PCR-product, a fusion of the attB-recombination- andΩ-leader-sequence could be synthesized. This fragment contains XhoI- anda NcoI-restriction sites at its termin. The PCR-product was isolated,digested and cloned into the XhoI- and NcoI-sites of plasmid plCH3441 inorder to replace the LoxP-Q-fusion. The intermediate plasmid was treatedwith KpnI and HindIII. By inserting this fragment into the correspondingsites of the binary vector BV10, a attB-sGFP-3′NTR-nos vector could beobtained which is transformable into Agrobacterium (plCH5151, see FIG.32).

A similar approach was used for cloning an analogous provector with thereporter gene GUS. The above described PCR-fragment was cloned into theXhoI- and NcoI-sites of the GUS-containing plasmid plCP3521 resulting inplasmid plCH4061. Finally, a KpnI-HindIII-fragment of plasmid plasmidplCH4061 was ligated into the binary-vector BV10, resulting in the finalvector plCH5161 which is capable of stable Agrobacterium-transformation(plCH5161, see FIG. 33).

C) Construction of a attB-Ubiquitin-Interferon-Containing 3′-Provector

In order to clone a fusion of a ubiquitin-sequence and interferon α2Binto an attB-3′-provector, both sequences were synthesized as a fusionand cloned into pUC19 (obtaining plasmid pUC5760). The vector wasdigested with NcoI and PstI and the resulting fragment was ligated intothe corresponding restriction-sites of plasmid plCH5781. The resultingplasmid plCH5951 contains an attB-recombination site, theubiquitin-interferon fusion, an 3′ nontranslated region, a nos promoterand a Kanamycin-expression-cassette. All said sequences are flanked byT-DNA borders (LB and RB, see FIG. 34).

D) In order to replace the 1-sequence by different IRES-elements whichcan serve as translational enhancers, a ClaI/ApaI-fragment was takenfrom plasmid plC1701 (that contains IRES_(mp)75(U1)) or plCH1731(contains IRES_(mp)75(cr)), respectively. The fragments were cloned intothe plasmid plCP5939 (similar to plasmid plCH5781, but withoutNcoI-restriction-site with ATG-sequence as transcriptioninitiation-codon). In the final binary vectors (plCH6871, plCP6891), theattb-recombinabon-site is located adjacent to an IRES-sequence of 75 bp(6871: U1-origin, 6891: CrTMV-origin), a sGFP-reporter gene and theregulatory elements 3′NTR and nos.

E) Cloning of Vector Type Polymerase-MP-LoxP into Binary Vectors

In order to clone the polymerase-MP carrying LoxP-provector into abinary vector, a KpnI/XhoI and a XhoI/HindIII-fragment were ligatedtogether into the vector plCBV10 which was digested with HindIII andKpnI. The resulting plasmid plCH4371 can be stably transformed intoAgrobacterium (FIG. 36).

F) Cloning of Vector Type LoxP-Gene of Interest-3′NTR-nos-terminatorinto Binary Vector

An analogous cloning strategy like in E) was used to clone theLoxP-reporter gene provector into a binary vector. Plasmid plCH3441 wasdigested with the enzymes KpnI and HindIII. The resulting fragment wasinserted in the corresponding sides of plCBV10 resulting in the binaryplasmid plCP4461 (see FIG. 37).

REFERENCE EXAMPLE 15

Delivery of Viral Vector Constructs by Infiltration of Agrobacteriumtumefaciens Suspension into Plant Leaves of N. benthamiana and N.tabacum

All provectors of the Cre/Lox and the integrase/att-system have beencloned into binary vectors which can be transformed stably intoAgrobacteria. Hence, DNA-delivery methods alternative to the describedtechniques are available.

A very simple delivery method is the infiltration of Agrobacteriasuspensions into intact leaves. This so called agroinfiltration wasinitially developed to analyze foreign gene expression and genesilencing in plants (Kaplia et al., 1997, Plant Science, 122, 101-108;Schöb et al., 1997, Mol. Gen. Genet., 256, 581-588). Agroinfiltration oftransgenic tobacco plants was performed according to a modified protocoldescribed by Yang et al., 2000, The Plant Journal, 22(6), 543-551.Agrobacterium tumefaciens strain GV3101 was transformed with individualconstructs (plCH4851, plCH5151, plCP1010, FIG. 15) was grown inLB-medium supplemented with Rifampicin 50 mg/l, carbencilin 50 mg/l and100 pM acetosyringone at 28° C. Agrobacterium cells of an overnightculture (5 ml) were collected by centrifugation (10 min, 4500 g) andresuspended in 10 mM MES (pH 5.5) buffer supplemented with 10 mM MgSO₄and 100 μM acetosyringone. The bacterial suspension was adjusted to afinal OD₆₀₀ of 0.8. In case of delivery of several constructs,Agrobacteria suspensions of different constructs were mixed in equalvolumes before infiltration.

Agroinfiltration was conducted on near fully expanded leaves that werestill attached to the intact plant. A bacterial suspension wasinfiltrated using a 5 ml syringe. By infiltrating 100 μl of bacterialsuspension into each spot (typically 34 cm² in infiltrated area), 8 to16 spots separated by veins could be arranged in a single tobacco leaf.After infiltration, plants were further grown under greenhouseconditions at 22° C. and 16 h light.

Leaves of N. benthamiana and N. tabacum that were infiltrated with theconstructs show expanding sectors of strong GFP-expression 8-12 daysafter infiltration. The expression could be detected under UV-lightdirectly on the infiltrated leaves. No GFP expression could be observedin case of controls (provector-combination without the integrase-clone).

REFERENCE EXAMPLE 16

Delivery of Viral Provectors by Infiltration of an Agrobacteriumtumefaciens Suspension into Plant Leaves of Transgenic Tobacco PlantsExpressing the Cre Recombinase (plCH1754)

Leaves of transgenic tobacco plants (transformed construct: plCH1754,species Nicotiana tabacum) infiltrated with construct plCP4371 andplCH4461 showed 16 days after infiltration growing sectors of strongGFP-expression which could be observed without microscope under UV-lighton intact plants. No GFP-expression was visible on wild type leavesinfiltrated with the same Agrobacteria suspension mix.

REFERENCE EXAMPLE 17

The use of IRES Sequences of Viral Origin (IRES_(mp)75 from CrTMV or U1)as Translational Enhancer Sequences

The presence of an att- or LoxP— recombination-site between promoter anda downstream located gene limits the efficiency of translation.Therefore, in most cases, it is necessary to use translational enhancingelements to reach an appropriative level of reporter gene-expression inthe provector-system. Cloning of the viral IRES sequencesIRES_(mp)75(U1) or IRES_(mp)75(cr) respectively between theattB-recombination site and GFP clearly increases the expression of thereportergene. Whereas plants, which have been infiltrated withGFP-containing provectors that carry no translational enhancer shownearly no detectable GFP-expression after 10 days, the use of saidIRES-sequences led to expression of GFP, which can be detected underUV-light without using a microscope after 5 days. The expression levelof gene of interest provided by IRES_(mp)75-based translational enhanceractivity is comparable or even higher than such provided by “omega”translational enhancer.

1. A process of producing a protein or polypeptide of interest in aplant or in plant cells, comprising: (i) transforming or transfecting aplant or plant cells with a nucleotide sequence having a coding regionencoding a fusion protein comprising the protein or polypeptide ofinterest, a signal peptide functional for targeting said fusion proteinto the apoplast, and a polypeptide capable of binding the fusion proteinto a cell wall component, (ii) enriching cell wall components havingexpressed and bound fusion protein, and (iii) separating the protein orpolypeptide of interest or a protein comprising the protein orpolypeptide of interest.
 2. The process of claim 1, wherein said plantis transfected for transient expression of said fusion protein.
 3. Theprocess of claim 1, wherein said plant is transfected using a viralvector.
 4. The process of claim 1, whereby a plant is grown up to adesired growth state before carrying out step (i).
 5. The process ofclaim 1, whereby said enriching of cell wall components of step (ii)comprises mechanical, physical or chemical separation of cell contents.6. The process of claim 1, wherein the polypeptide capable of bindingthe fusion protein to a cell wall component binds to a plant cell wallpolysaccharide.
 7. The process according to claim 6, wherein thepolypeptide capable of binding the fusion protein to a cell wallcomponent binds to β-1,4-glycan.
 8. The process according to claim 6,wherein the polypeptide capable of binding the fusion protein to a cellwall component binds to cellulose.
 9. The process according to claim 6,wherein the polypeptide capable of binding the fusion protein to a cellwall component binds to hemicellulose.
 10. The process according toclaim 6, wherein the polypeptide capable of binding the fusion proteinto a cell wall component binds to pectin.
 11. The process according toclaim 1, wherein said fusion protein comprises a pectin methylesteraseor a functional fragment thereof which functions as said signal peptideand as said polypeptide capable of binding said fusion protein to a cellwall component.
 12. The process according to claim 1, wherein saidfusion protein comprises the cysteine-rich domain of the tobacco NtTLPRprotein or a functional fragment thereof capable of cross-linking saidfusion protein to a cell wall component.
 13. The process according toclaim 1, further comprising cleaving said fusion protein for obtainingsaid protein or polypeptide of interest free of said signal peptideand/or said polypeptide capable of binding said fusion protein to a cellwall component.
 14. The process of claim 13, wherein said fusion proteincomprises a cleavage sequence allowing cleavage of the fusion protein.15. The process of claim 14, wherein the cleavage sequence is recognizedby a site-specific protease.
 16. The process of claim 15, wherein thecleavage sequence comprises the C-terminal portion of ubiquitin.
 17. Theprocess according to claim 1, wherein said fusion protein contains saidsignal peptide at its N-terminal end followed by said polypeptidecapable of binding the fusion protein to a cell wall component followedby said protein or polypeptide of interest.
 18. The process according toclaim 1, wherein said fusion protein contains said signal peptide at itsN-terminal end followed by said protein or polypeptide of interest whichis followed by said polypeptide capable of binding the fusion protein toa cell wall component.
 19. The process according to claim 1, whereinsaid fusion protein contains said signal peptide at its N-terminal endand said protein or polypeptide of interest is flanked on both sides bya polypeptide capable of binding the fusion protein to a cell wallcomponent.
 20. The process claim 1, wherein said nucleotide sequence fortransforming or transfecting a plant or plant cells is DNA.
 21. Theprocess of claim 1, wherein said nucleotide sequence for transforming ortransfecting a plant or plant cells is RNA.
 22. The process according toclaim 1, wherein said protein or polypeptide of interest contains one ormore affinity peptide tags.
 23. The process of claim 22, wherein saidaffinity peptide tag is selected from the group consisting of anoleosinprotein or part thereof, an intein or part thereof, an additional cellwall binding domain, a starch binding domain, a streptavidin epitope-tagsequence, glutathione-S-transferase (GST) affinity tag, a 6×His affinitytag, and a calmodulin binding peptide (CBP) affinity tag.
 24. Theprocess of claim 1, whereby said nucleotide sequence is amplified and/orexpressed in said plant by a process comprising: providing the plant orcells thereof with at least one precursor vector designed for undergoingprocessing in cells of said plant, whereby due to said processing saidplant cell is endowed with at least one replicon which provides for saidamplification and/or expression.
 25. The process of claim 24, whereinthe plant or cells thereof are provided with at least two precursorvectors.
 26. The process of claim 24, wherein due to said processingsaid plant cell is endowed with at least two replions which (a) arestructurally related to each other owing to said processing; (b) arefunctionally distinct from each other; and (c) provide for saidamplification and/or expression.
 27. A vector comprising said nucleotidesequence encoding said fusion protein as defined in claim
 1. 28. Plantcells or plants transformed or transfected with said nucleotide sequenceencoding said fusion protein as defined in claim
 1. 29. A protein ofinterest or fusion protein obtained by the process of claim 1.